Note: Descriptions are shown in the official language in which they were submitted.
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INTRACELLULAR SIGNALING MOLECULES
TECHNICAL FIELD
The invention relates to novel nucleic acids, intracellular signaling
molecules encoded by these
nucleic acids, and to the use of these nucleic acids and proteins in the
diagnosis, treatment, and
prevention of cell proliferative, endocrine, autoimmunelinflammatory,
neurological, gastrointestinal,
reproductive, developmental, and vesicle trafficking disorders. The invention
also relates to the
assessment of the effects of exogenous compounds on the expression of nucleic
acids and intracellular
signaling molecules.
BACKGROUND OF THE INVENTION
Cell-cell communication is essential for the growth, development, and survival
of multicellular
organisms. Cells communicate by sending and receiving molecular signals. An
example of a
molecular signal is a growth factor, which binds and activates a specific
transmembrane receptor on
the surface of a target cell. The activated receptor transduces the signal
intracellularly, thus initiating
a cascade of biochemical reactions that ultimately affect gene transcription
and cell cycle progression
in the target cell.
Intracellular signaling is the process by which cells respond to extracellular
signals (hormones,
neurotransmitters, growth and differentiation factors, etc.) through a cascade
of biochemical reactions
that begins with the binding of a signaling molecule to a cell membrane
receptor and ends with the
activation of an intracellular target molecule. Intermediate steps in the
process involve the activation
of various cytoplasmic proteins by phosphorylation via protein kinases, and
their deactivation by protein
phosphatases, and the eventual translocation of some of these activated
proteins to the cell nucleus
where the transcription of specific genes is triggered. The intracellular
signaling process regulates all
types of cell functions including cell proliferation, cell differentiation,
and gene transcription, and
involves a diversity of molecules including protein kinases and phosphatases,
and second messenger
molecules such as cyclic nucleotides, calcium-calmodulin, inositol, and
various mitogens that regulate
protein phosphorylation.
A distinctive class of signal transduction molecules are involved in odorant
detection. The
process of odorant detection involves speci~.c recognition by odorant
receptors. The olfactory mucosa
also appears to possess an additional group of odorant binding proteins which
recognize and bind
separate classes of odorants. For example, cDNA clones from rat have been
isolated which
correspond to mRNAs highly expressed in olfactory mucosa but not detected in
other tissues. The
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proteins encoded by these clones are homologous to proteins that bind
lipopolysaccharides or
polychlorinated biphenyls, and the different proteins appear to be expressed
in specific areas of the
mucosal tissue. These proteins are believed to interact with odorants before
or after specific
recognition by odorant receptors, perhaps acting as selective signal filters
(Dear, T.N. et al. (1991)
EMBO J. 10:2813-2819; Vogt, R.G. et al. (1991) J. Neurobiol. 22:74-84).
Cells also respond to changing conditions by switching off signals. Many
signal transduction
proteins are short-lived and rapidly targeted for degradation by covalent
ligation to ubiquitin, a highly
conserved small protein. Cells also maintain mechanisms to monitor changes in
the concentration of
denatured or unfolded proteins in membrane-bound extracytoplasmic
compartments, including a
transmembrane receptor that monitors the concentration of available chaperone
molecules in the
endoplasmic reticulum and transmits a signal to the cytosol to activate the
trauscription of nuclear
genes encoding chaperones in the endoplasmic reticulum.
Certain proteins in intracellular signaling pathways serve to link or cluster
other proteins
involved in the signaling cascade. These proteins are referred to as scaffold,
anchoring, or adaptor
proteins. (For review, see Pawson, T. and J.D. Scott (1997) Science 278:2075-
2080.) As many
intracellular signaling proteins such as protein kinases and phosphatases have
relatively broad
substrate specificities, the adaptors help to organize the component signaling
proteins into specific
biochemical pathways. Many of the above signaling molecules are characterized
by the presence of
particular domains that promote protein-protein interactions. A sampling of
these domains is discussed
below, along with other important intracellular messengers.
Intracellular Signaling Second Messenger Molecules
Protein Phos hor 1
Protein kinases and phosphatases play a key role in the intracellular
signaling process by
controlling the phosphorylation and activation of various signaling proteins.
The high energy phosphate
for this reaction is generally transferred from the adenosine triphosphate
molecule (ATP) to a
particular protein by a protein kinase and removed from that protein by a
protein phosphatase. Protein
kinases are roughly divided into two groups: those that phosphorylate serine
or threonine residues
(serine/threonine kinases, STK) and those that phosphorylate tyrosine residues
(protein tyrosine
kinases, PTK). A few protein kinases have dual specificity for
serine/threonine and tyrosine residues.
Almost all kinases contain a conserved 250-300 amino acid catalytic domain
containing specific
residues and sequence motifs characteristic of the kinase family (Hardie, G.
and S. Hanks (1995) The
Protein Kinase Facts Books, Vol I:7-20, Academic Press, San Diego, CA).
STKs include the second messenger dependent protein kinases such as the cyclic-
AMP
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dependent protein kinases (PKA), involved in mediating hormone-induced
cellular responses;
calcium-calinodulin (CaM) dependent protein kinases, involved in regulation of
smooth muscle
contraction, glycogen breakdown, and neurotransmission; and the mitogen-
activated protein kinases
(MAP kinases) which mediate signal transduction from the cell surface to the
nucleus via
phosphorylation cascades. Altered PKA expression is implicated in a variety of
disorders and
diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and
cardiovascular disease
(Isselbacher, K.J. et al. (1994) Harrison's Principles of Internal Medicine,
McGraw-Hill, New York,
NYC pp. 416-431, 1887).
PTKs are divided into transmembrane, receptor PTKs and nontransmembrane, non-
receptor
PTKs. Transmembrane PTKs are receptors for most growth factors. Non-receptor
PTKs lack
transmembrane regions and, instead, form complexes with the intracellular
regions of cell surface
receptors. Receptors that function through non-receptor PTKs include those for
cytokines and
hormones (growth hormone and prolactin) and antigen-specific receptors on T
and B lymphocytes.
Many of these PTKs were first identified as the products of mutant oncogenes
in cancer cells in
which their activation was no longer subject to normal cellular controls. In
fact, about one third of the
known oncogenes encode PTKs, and it is well known that cellular transformation
(oncogenesis) is
often accompanied by increased tyrosine phosphorylation activity (Gharbonneau
H. and N.K. Tonks
(1992) Annu. Rev. Cell Biol. 8:463-493).
An additional family of protein kinases previously thought to exist only in
prokaryotes is the
histidine protein kinase family (HPK). HPKs bear little homology with
mammalian STKs or PTKs but
have distinctive sequence motifs of their own (Davie, J.R. et al. (1995) J.
Biol. Chem.
270:19861-19867). A histidine residue in the N-terminal half of the molecule
(region I) is an
autophosphorylation site. Three additional motifs located in the C-terminal
half of the molecule include
an invariant asparagine residue in region lI and two glycine-rich loops
characteristic of nucleotide
binding domains in regions III and IV. Recently a branched chain alpha-
ketoacid dehydrogenase
kinase has been found with characteristics of HPK in rat (Davie et al.,
supra).
Protein phosphatases regulate the effects of protein kinases by removing
phosphate groups
from molecules previously activated by kinases. The two principal categories
of protein phosphatases
are the protein (serine/threonine) phosphatases (PPs) and the protein tyrosine
phosphatases (PTPs).
PPs dephosphorylate phosphoserine/threonine residues and are important
regulators of many
CAMP-mediated hormone responses (Cohen, P. (1989) Annu. Rev. Biochem. 58:453-
508). PTPs
reverse the effects of protein tyrosine kinases and play a significant role in
cell cycle and cell signaling
processes (Charbonneau and Tonks, supra). As previously noted, many PTKs are
encoded by
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oncogenes, and oncogenesis is often accompanied by increased tyrosine
phosphorylation activity. It is
therefore possible that PTPs may prevent or reverse cell transformation and
the growth of various
cancers by controlling the levels of tyrosine phosphorylation in cells. This
hypothesis is supported by
studies showing that overexpression of PTPs can suppress transformation in
cells, and that specific
inhibition of PTPs can enhance cell transformation (Charbonneau and Tonks,
supt-a).
Phospholipid and Inositol-phosphate Sag
Inositol phospholipids (phosphoinositides) are involved in an intracellular
signaling pathway that
begins with binding of a signaling molecule to a G-protein linked receptor in
the plasma membrane.
This leads to the phosphorylation of phosphatidylinositol (PI) residues on the
inner side of the plasma
membrane to the biphosphate state (P1P2) by inositol kinases. Simultaneously,
the G-protein linked
receptor binding stimulates a trimeric G-protein which in turn activates a
phosphoinositide-specific
phospholipase C-~3. Phospholipase C-~3 then cleaves P71'2 into two products,
inositol triphosphate (1P3)
and diacylglycerol. These two products act as mediators for separate signaling
events. IP3 diffuses
through the plasma membrane to induce calcium release from the endoplasmic
reticulum (ER), while
diacylglycerol remains in the membrane and helps activate protein kinase C, a
serine-threonine kinase
that phosphorylates selected proteins in the target cell. The calcium response
initiated by 1P3 is
terminated by the dephosphorylation of IP3 by specific inositol phosphatases.
Cellular responses that
are mediated by this pathway are glycogen breakdown in the liver in response
to vasopressin, smooth
muscle contraction in response to acetylcholine, and thrombin-induced platelet
aggregation.
Inositol-phosphate signaling controls tubby, a membrane bound transcriptional
regulator that
serves as an intracellular messenger of Gccq coupled receptors (Santagata et
al. (2001) Scienee
292:2041-2050). Members of the tubby family contain a C-terminal tubby domain
of about 260 amino
acids that binds to double-stranded DNA and an N-terminal transcriptional
activation domain. Tubby
binds to phosphatidylinositol 4,5-bisphosphate, which localizes tubby to the
plasma membrane.
Activation of the G-protein ocq leads to activation of phospholipase C-(3 and
hydrolysis of
phosphoinositide. Loss of phosphatidylinositol 4,5-bisphosphate causes tubby
to dissociate from the
plasma membrane and to trauslocate to the nucleus where tubby regulates
transcription of its target
genes. Defects in the tubby gene are associated with obesity, retinal
degeneration, and hearing loss
(Boggon, T.J. et al. (1999) Science 286:2119-2125).
Cyclic Nucleotide Si~nalix~
Cyclic nucleotides (CAMP and cGMP) function as intracellular second messengers
to
transduce a variety of extracellular signals including hormones, light, and
neurotransmitters. In
particular, cyclic-AMP dependent protein kinases (PKA) are thought to account
for all of the effects
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of cAMP in most mammalian cells, including various hormone-induced cellular
responses. Visual
excitation and the'phototransmission of light signals in the eye is controlled
by cyclic-GMP regulated,
Caa+-specific channels. Because of the importance of cellular levels of cyclic
nucleotides in mediating
these various responses, regulating the synthesis and breakdown of cyclic
nucleotides is an important
matter. Thus adenylyl cyclase, which synthesizes cAMP from AMP, is activated
to increase cAMP
levels in muscle by binding of adrenaline to (3-adrenergic receptors, while
activation of guanylate
cyclase and increased cGMP levels in photoreceptors leads to reopening of the
Ca2+-specific channels
and recovery of the dark state in-the eye. There are nine known transmembrane
isoforms of
mammalian adenylyl cyclase, as well as a soluble form preferentially expressed
in testis. Soluble
1o adenylyl cyclase contains a P-loop, or nucleotide binding domain, and may
be involved in male fertility
(Buck, J. et al. (1999) Proc. Natl. Acad. Sci. USA 96:79-84).
In contrast, hydrolysis of cyclic nucleotides by CAMP and cGMP-specific
phosphodiesterases
(PDEs) produces the opposite of these and other effects mediated by increased
cyclic nucleotide
levels. PDEs appear to be particularly important in the regulation of cyclic
nucleotides, considering
the diversity found in this family of proteins. At least seven families of
mammalian PDEs (PDE1-7)
have been identified based on substrate specificity and affinity, sensitivity
to cofactors, and sensitivity
to inhibitory drugs (Beavo, J.A. (1995) Physiol. Rev. 75:725-748). PDE
inhibitors have been found to
be particularly useful in treating various clinical disorders. Rolipram, a
specific inhibitor of PDE4, has
been used in the treatment of depression, and similar inhibitors are
undergoing evaluation as
anti-inflammatory agents. Theophylline is a nonspecific PDE inhibitor used in
the treatment of
bronchial asthma and other respiratory diseases (Banner, K.H. and C.P. Page
(1995) Eur. Respir. J.
8:996-1000).
Calcium Si~nalina Molecules
Ca2+ is another second messenger molecule that is even more widely used as an
intracellular
mediator than cAMP. Ca2+ can enter the cytosol by two pathways, in response to
extracellular
signals. One pathway acts primarily in nerve signal transduction where Ca~+
enters a nerve terminal
through a voltage-gated Caa+ channel. The second is a more ubiquitous pathway
in which Ca2+ is
released from the ER into the cytosol in response to binding of an
extracellular signaling molecule to a
receptor. Ca2+ directly activates regulatory enzymes, such as protein kinase
C, which trigger signal
transduction pathways. Ca2+ also binds to specific Caa+-binding proteins
(CBPs) such as calmodulin
(CaM) which then activate multiple target proteins in the cell including
enzymes, membrane transport
pumps, and ion channels. CaM interactions are involved in a multitude of
cellular processes including,
but not limited to, gene regulation, DNA synthesis, cell cycle progression,
mitosis, cytokinesis,
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cytoskeletal organization, muscle contraction, signal transduction, ion
homeostasis, exocytosis, and
metabolic regulation (Cello, M.R. et al. (1996) Guidebook to Calcium-binding
Proteins, Oxford
University Press, Oxford, UK, pp. 15-20). Some Ca2+ binding proteins are
characterized by the
presence of one or more EF-hand Ca2+ binding motifs, which are comprised of 12
amino acids flanked
by o~,-helices (Cello, supra). The regulation of CBPs has implications for the
control of a variety of
disorders. Calcineurin, a CaM-regulated protein phosphatase, is a target for
inhibition by the
i_mmunosuppressive agents cyclosporin and FK506. This indicates the importance
of calcineurin and
CaM in the immune response and immune disorders (Schwaninger M. et al. (1993)
J. Biol Chem.
268:23111-23115). The level of CaM is increased several-fold in tumors and
tumor-derived cell lines
1o for various types of cancer (Rasmussen, C.D. and A.R. Means (1989) Trends
Neurosci. 12:433-438).
The annexins are a family of calcium-binding proteius that associate with the
cell membrane
(Towle, C.A. and B.V. Treadwell (1992) J. Biol. Chem. 267:5416-5423). Annexins
reversibly bind to
negatively charged phospholipids (phosphatidylcholine and phosphatidylserine)
in a calcium dependent
manner. Annexins participate in various processes pertaining to signal
transduction at the plasma
membrane, including membrane-cytoskeleton interactions, phospholipase
inhibition, anticoagulation,
and membrane fusion. Annexins contain four to eight repeated segments of about
60 residues. Each
repeat folds into five alpha helices wound into a right-handed superhelix.
G-Protein. Signaling
Guanine nucleotide binding proteins (G-proteins) are critical mediators of
signal trausduction
between a particular class of extracellular receptors, the G-protein coupled
receptors (GPCRs), and
intracellular second messengers such as CAMP and Ca2+. G-proteins are linked
to the cytosolic side
of a GPCR such that activation of the GPCR by ligand binding stimulates
binding of the G-protein to
GTP, inducing an "active" state in the G-protein. In the active state, the G-
protein acts as a signal to
trigger other events in the cell such as the increase of CAMP levels or the
release of Ca2+ into the
cytosol from the ER, which, in turn, regulate phosphorylation and activation
of other intracellular
proteins. Recycling of the G-protein to the inactive state involves hydrolysis
of the bound GTP to
GDP by a GTPase activity in the G-protein. (See Alberts, B. et al. (1994)
Molecular Biolog o~ f the
Cell Garlaud Publishing, Inc. New York,- 1VY, pp.734-759.) The superfamily of
G-proteins consists of
several families which may be grouped as translational factors, heterotrimeric
G-proteins involved iu
transmembrane signaling processes, and low molecular weight (LMW) G-proteius
including the proto-
oncogene Ras proteins and products of rab, rap, rho, rac, smg2l, smg25, YPT,
SEC4, and ARF genes,
and tubulins (Kaziro, Y. et al. (1991) Annu. Rev. Biochem. 60:349-400). In.
all cases, the GTPase
activity is regulated through interactions with other proteins. G protein
activity is triggered by seven-
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transmembrane cell surface receptors (G-protein coupled receptors) which
respond to lipid analogs,
amino acids and their derivatives, peptides, cytokines, and specialized
stimuli such as light, taste, and
odor. Activation of the receptor by its stimulus causes the replacement of the
G protein-bound GDP
with GTP. Ga-GTP dissociates from the receptor/(3y complex, and each of these
separated
components can interact with and regulate downstream effectors. The signaling
stops when Goc
hydrolyzes its bound GTP to GDP and reassociates with the (3'y complex (Neer,
supra).
Ras proteins are membrane-associated molecular switches that bind GTP and GDP
and
slowly hydrolyze GTP to GDP. This intrinsic GTPase activity of ras is
stimulated by a family of
proteins collectively known as 'GAP' or GTPase-activating proteins. Since the
GTP bound form of
ras is active, ras-GAP proteins down-regulate ras. ras Gap is an alpha-helical
domain that accelerates
the GTPase activity of Ras, thereby "switching" it into an "off" position
(Wittinghofer, A. et al. (1997)
FEBS Lett. 410:63-67)
Guanine nucleotide binding proteins (GTP-binding proteins) participate in a
wide range of
regulatory functions in all eukaryotic cells, including metabolism, cellular
growth, differentiation, signal
transduction, cytoskeletal organization, and intracellular vesicle transport
and secretion. In higher
organisms they are involved in signaling that regulates such processes as the
immune response
(Aussel, C. et al. (1988) J. Tmmunol. 140:215-220), apoptosis,
differentiation, and cell proliferation
including oncogenesis (Dhanasekaran, N. et al. (1998) Oncogene 17:1383-1394).
Exchange of bound
GDP for GTP followed by hydrolysis of GTP to GDP provides the energy that
enables GTP-binding
proteins to alter their conformation and interact with other cellular
components. The superfamily of
GTP-binding proteins consists of several families and may be grouped as
translational factors,
heterotrimeric GTP-binding proteins involved in transmembrane signaling
processes (also called G-
proteins), and low molecular weight (LMW) GTP-binding proteins including the
proto-oncogene Ras
proteins and products of rab, rap, rho, rac, smg2l, smg25, YPT, SEC4, and ARF
genes, and tubulins
(I~aziro, Y. et al. (1991) Annu. Rev. Biochem. 60:349-400). In all cases, the
GTPase activity is
regulated through interactions with other proteins.
The low molecular weight (LMW) GTP-binding proteins regulate cell growth, cell
cycle
control, protein secretion, and intracellular vesicle interaction. These GTP-
binding proteins respond to
extracellular signals from receptors and activating proteins by transducing
mitogenic signals (Tavitian,
3o A. (1995) C. R. Seances Soc. Biol. Fil. 189:7-12). Low molecular weight GTP-
binding proteins
consist of single polypeptides of 21-30kD which are able to bind to and
hydrolyze GTP, thus cycling
from an inactive to an active state.
Low molecular weight GTP-binding proteins play critical roles in cellular
protein trafficking
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events, such as the translocation of proteins and soluble complexes from the
cytosol to the membrane
through an exchange of GDP for GTP (Ktistakis, N.T. (1998) BioEssays 20:495-
504). In vesicle
transport, the interaction between vesicle- and target- specific identifiers
(v-SNARES and tSNAREs)
docks the vesicle to the acceptor membrane. The budding process is regulated
by GTPases such as
the closely related ADP ribosylation factors (ARFs) and SAR proteins, while
GTPases such as Rab
allow assembly of SNARE complexes and may play a role in removal of defective
complexes
(Rothman, J.E. anal F.T. Wieland (1996) Science 272:227-234). The rab proteins
control the
translocation of vesicles to and from membranes for-protein localization,
protein processing, and
secretion. The rho GTP-binding proteins control signal transduction pathways
that liuk growth factor
receptors to actin polymerization which is necessary for normal cellular
growth and division. The ran
GTP binding proteins are located in the nucleus of cells and have a key role
in nuclear protein import,
the control of DNA synthesis, and cell-cycle progression (Hall, A. (1990)
Science 249:635-640;
Scheffzek, K. et al. (1995) Nature 374:378-381).
The cycling of LMW GTP-binding proteins between the GTP-bound active form and
the
GDP-bound inactive form is regulated by additional proteins. Guanosine
nucleotide exchange factors
(GEFs) increase the rate of nucleotide dissociation by several orders of
magnitude, thus facilitating
release of GDP and loading with GTP. Certain Ras-family proteins are also
regulated by guanine
nucleotide dissociation inhibitors (GDIs), which inhibit GDP dissociation. The
intrinsic rate of GTP
hydrolysis of the LMW GTP-binding proteins is typically very slow, but it can
be stimulated by several
orders of magnitude by GTPase-activating proteins (GAPS) (Geyer, M. and
Wittinghofer, A. (1997)
C~rr. Opin. Struct. Biol. 7:786-792).
Heterotrimeric G=proteins are composed of 3 subunits, a, (3, and y, which in
their inactive
conformation associate as a trimer at the inner face of the plasma membrane.
Gcc binds GDP or GTP
and contains the GTPase activity. The (3y complex enhances binding of Goc to a
receptor. G'y is
necessary for the folding and activity of G(3 (Veer, E.J. et al. (1994) Nature
371:297-300). Multiple
homologs of each subunit have been identified in mammalian tissues, and
different combinations of
subunits have specific functions and tissue specificities (Spiegel, A.M.
(1997) J. Inher. Metab. Dis.
20:113-121). The (3 subunits, also known as G-(3 proteins or (3 transducins,
contain seven tandem
repeats of the WD-repeat sequence motif, a motif found in many proteins with
regulatory functions.
Mutations and variant expression of (3 transducin proteins are linked with
various disorders (Neer, E.J.
et al. (1994) Nature 371:297-300; Margottin, F. et al. (1998) Mol. Cell. 1:565-
574).
The alpha subunits of heterotrimeric G-proteins can be divided into four
distinct classes. The
cc-s class is sensitive to ADP-ribosylation by pertussis toxin which uncouples
the receptor:G-protein
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interaction. This uncoupling blocks signal transduction to receptors that
decrease cAMP levels which
normally regulate ion channels and activate phospholipases. The inhibitory ot-
I class is also susceptible
to modification by pertussis toxin which prevents cc-I from lowering CAMP
levels. Two novel classes
of a subunits refractory to pertussis toxin modification are oc-q, which
activates phospholipase C, and
cc-12, which has sequence homology with the Dt-osophila gene cofieerti~ta and
may contribute to the
regulation of embryonic development (Simon, M.I. (1991) Science 252:802-808).
The mammalian G[3 and Gy subunits, each about 340 amino acids long, share more
than 80%
homology. The G(3 subunit (also called transducin) contains seven repeating
units, each about 43
amino acids long. The activity of both subunits may be regulated by other
proteins such as calmodulin
and phosducin or the neural protein GAP 43 (Clapham, D. and E. Neer (1993)
Nature 365:403-406).
The (3 and 'y subunits are tightly associated. The (3 subunit sequences are
highly conserved between
species, implying that they perform a fundamentally important role in the
organization and function of
G-protein linked systems (Van der Voorn, L. (1992) FEBS Lett. 307:131-134).
They contain seven
tandem repeats of the WD-repeat sequence motif, a motif found in many proteins
with regulatory
functions. WD-repeat proteins contain from four to eight copies of a loosely
conserved repeat of
approximately 40 amino acids which participates in protein-protein
interactions. Mutations and variant
expression of (3 transducin proteins are linked with various disorders.
Mutations in LIS 1, a subunit of
the human platelet activating factor acetylhydrolase, cause Miller-Dieker
lissencephaly. RACK1
binds activated protein kinase C, and RbAp48 binds retinoblastoma protein.
CstF is required for
polyadenylation of mammalian pre-mRNA ih vitf~o and associates with subunits
of cleavage-
stimulating factor. Defects in the regulation of (3-catenin contribute to the
neoplastic transformation of
human cells. The WD40 repeats of the human F-box protein bTrCP mediate binding
to (3-catenin,
thus regulating the targeted degradation of [3-catenin by ubiquitin ligase
(Neer et al., supra; Hart, M.
et al. (1999) Curr. Biol. 9:207-210). The 7 subunit primary structures are
more variable than those of
the (3 subunits. They are often post-translationally modified by
isoprenylation and carboxyl-methylation
of a cysteine residue four amino acids from the C-terminus; this appears to be
necessary for the
interaction of the ~3'y subunit with the membrane and with other G-proteins.
The [37 subunit has been
shown to modulate the activity of isoforms of adenylyl cyclase, phospholipase
C, and some ion
channels. It is involved in receptor phosphorylation via specific kinases, and
has been implicated in the
3o p2lras-dependent activation of the MAP kinase cascade and the recognition
of specific receptors by
G-proteins (Clapham and Neer, supra).
G-proteins interact with a variety of effectors including adenylyl cyclase
(Clapham and Neer,
supt~a). The signaling pathway mediated by cAMP is mitogenic in hormone-
dependent endocrine
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tissues such as adrenal cortex, thyroid, ovary, pituitary, and testes. Cancers
in these tissues have been
related to a mutationally activated form of a Ga s known as the gsp (Gs
protein) oncogene
(Dhanasekaran, N. et al. (1998) Oncogene 17:1383-1394). Another effector is
phosducin, a retinal
phosphoprotein, which forms a specific complex with retinal G(3 and Gy (G(3y)
and modulates the
ability of G[3~y to interact with retinal Ga (Clapham and Neer, supf~a).
Irregularities in the G-protein signaling cascade may result in abnormal
activation of
leukocytes and lymphocytes, leading to the tissue damage and.destruction seen
in many inflammatory
and autoimmune diseases such as rheumatoid arthritis, biliary cirrhosis,
hemolytic anemia, lupus
erythematosus, and thyroiditis. Abnormal cell proliferation, including cyclic
AMP stimulation of brain,
thyroid, adrenal, and gonadal tissue proliferation is regulated by G proteins.
Mutations in Got subunits
have been found in. growth-hormone-secreting pituitary somatotroph tumors,
hyperfunctioning thyroid
adenomas, and ovarian and adrenal neoplasms (Meij, J.T.A. (1996) Mol. Cell
Biochem. 157:31-38;
Aussel, C. et al. (1988) J. Tm_m__unol. 140:215-220).
LMW G-proteins are GTPases which regulate cell growth, cell cycle control,
protein
secretion, and intracellular vesicle interaction. They consist of single
polypeptides which, like the alpha
subunit of the heterotrimeric G-proteins, are able to bind to and hydrolyze
GTP, thus cycling between
an inactive and an active state. LMW G-proteins respond to extracellular
signals from receptors and
activating proteins by transducing mitogenic signals involved in various cell
functions. The binding and
hydrolysis of GTP regulates the response of LMW G-proteins and acts as an
energy source during
this process (Bokoeh, G.M. and C.J. Der (1993) FASEB J. 7:750-759).
At least sixty members of the LMW G-protein superfamily have been identified
and are
currently grouped into the ras, rho, arf, sarl, ran, and rab subfamilies.
Activated ras genes were
initially found in human cancers, and subsequent studies confrtmed that ras
function is critical in
determining whether cells continue to grow or become differentiated. Ras1 and
Ras2 proteins
stimulate adenylate cyclase (Kaziro et al., supf-a), affecting a broad array
of cellular processes.
Stimulation of cell surface receptors activates Ras which, in turn, activates
cytoplasmic kinases. These
kinases translocate to the nucleus and activate key transcription factors that
control gene expression
and protein synthesis (Barbacid, M. (1987) Annu. Rev. Biochem. 56:779-827;
Treisman, R. (1994)
C~rr. Opin. Genet. Dev. 4:96-98). Other members of the LMW G-protein
superfamilyhave roles in
3o signal transduction that vary with the function of the activated genes and
the locations of the G-
proteins that initiate the activity. Rho G-proteins control signal
transduction pathways that link growth
factor receptors to actin polymerization, which is necessary for normal
cellular growth and division.
The rab, arf, and sarl families of proteins control the translocation of
vesicles to and from membranes
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for protein processing, localization, and secretion. Vesicle- and target-
specific identifiers (v-SNARES
and t-SNARES) bind to each other and dock the vesicle to the acceptor
membrane. The budding
process is regulated by the closely related ADP ribosylation factors (ARFs)
and SAR proteins, while
rab proteins allow assembly of SNARE complexes and may play a role in removal
of defective
complexes (Rothman, J. and F. Wieland (1996) Science 272:227-234). Ran G-
proteins are located in
the nucleus of cells and have a key role in nuclear protein import, the
control of DNA synthesis, and
cell-cycle progression (Hall, A. (1990) Science 249:635-640; Barbacid, supt-a;
Ktistakis, N. (1998)
BioEssays 20:495-504; and Sasaki, T. and Y. Takai (1998) Biochem. Biophys.
Res. Commun.
245:641-645).
The function of Rab proteins in vesicular transport requires the cooperation
of many other
proteins. Specifically, the membrane-targeting process is assisted by a series
of escort proteins
(Khosravi-Far, R. et al. (1991) Proc. Natl. Aced. Sci. USA 88:6264-6268). In
the medial Golgi, it has
been shown that GTP bound Rab proteins initiate the binding of VAMP-like
proteins of the transport
vesicle to syntaxin-like proteins on the acceptor membrane, which subsequently
triggers a cascade of
protein-binding and membrane-fusion events. After transport, GTPase-activating
proteins (GAPS) in
the target membrane are responsible for converting the GTP-bound Rab proteins
to their GDP-bound
state. And finally, guanine-nucleotide dissociation inhibitor (GDI) recruits
the GDP-bound proteins to
their membrane of origin.
The cycling of LMW G-proteins between the GTP-bound active form and the GDP-
bound
inactive form is regulated by a variety of proteins. Guanosine nucleotide
exchange factors (GEFs)
increase the rate of nucleotide dissociation by several orders of magnitude,
thus facilitating release of
GDP and loading with GTP. The best characterized is the mammalian homolog of
the Drosophila
Son-of Sevenless protein. Certain Ras-family proteins are also regulated by
guanine nucleotide
dissociation inhibitors (GDIs), which inhibit GDP dissociation. The intrinsic
rate of GTP hydrolysis of
the LMW G-proteins is typically very slow, but it can be stimulated by several
orders of magnitude by
GTPase-activating proteins (GAPS) (Geyer, M. and A. Wittinghofer (1997) C~rr.
Opin. Struct. Biol.
7:786-792). Both GEF and GAP activity may be controlled in response to
extracellular stimuli and
modulated by accessory proteins such as RalBP1 and POB 1. Mutant Ras-family
proteins, which bind
but cannot hydrolyze GTP, are permanently activated, and cause cell
proliferation or cancer, as do
GEFs that inappropriately activate LMW G-proteins, such as the human oncogene
NET1, a Rho-GEF
(Drives, G.T. et al. (1990) Mol. Cell Biol. 10:1793-1798; Alberts, A.S. and R.
Treisman (1998) EMBO
J. 14:4075-4085).
A member of the ARF family of G-proteins is centaurin beta 1A, a regulator of
membrane
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traffic and the actin cytoskeleton. The centaurin (3 family of GTPase-
activating proteins (GAPS) and
Arf guanine nucleotide exchange factors contain pleckstrin homology (PH)
domains which are
activated by phosphoinositides. PH domains bind phosphoinositides, implicating
PH domains in
signaling processes. Phosphoinositides have a role in converting Arf GTP to
Arf GDP via the
centaurin /3 family and a role in Arf activation (Kam, J.L. et al. (2000) J.
Biol. Chem. 275:9653-9663).
The rho GAP family is also implicated in the regulation of actin
polymerization at the plasma
membrane and in several cellular processes. The gene ARHGAP6 encodes GTPase-
activating
protein 6 isoform 4. Mutations in ARHGAP6, seen as a deletion of a S00 kb
critical region in Xp22.3,
causes the syndrome microphthalmia with linear skin defects (MLS). MLS is an X-
linked dominant,
male-lethal syndrome (Prakash, S.K. et al. (2000) Hum. Mol. Genet. 9:477-488).
Rab proteins have a highly variable amino terminus containing membrane-
specific signal
information and a prenylated carboxy terminus which determines the target
membrane to which the
Rab proteins anchor. More than 30 Rab proteins have been identified in a
variety of species, and each
has a characteristic intracellular location and distinct transport function.
In particular, Rab1 anal Rab2
are important in ER-to-Golgi transport; Rab3 transports secretory vesicles to
the extracellular
membrane; RabS is localized to endosomes and regulates the fusion of early
endosomes into late
endosomes; Rab6 is specific to the Golgi apparatus and regulates infra-Golgi
transport events; Rab7
and Rab9 stimulate the fusion of late endosomes and Golgi vesicles with
lysosomes, respectively; and
RablO mediates vesicle fusion from the medial Golgi to the traps Golgi. Mutant
forms of Rab proteins
are able to block protein transport along a given pathway or alter the sizes
of entire organelles.
Therefore, Rabs play key regulatory roles in membrane trafficking
(Schimmoller, LS. and S.R. Pfeffer
(1998) J. Biol. Chem. 243:22161-22164).
The function of Rab proteins in vesicular transport requires the cooperation
of many other
proteins. Specifically, the membrane-targeting process is assisted by a series
of escort proteins
(Khosravi-Far, R. et al. (1991) Proc. Natl. Acad. Sci. USA 88:6264-6268). In
the medial Golgi, it has
been shown that GTP-bound Rab proteins initiate the binding of VAMP-like
proteins of the transport
vesicle to syntaxin-like proteins on the acceptor membrane, which subsequently
triggers a cascade of
protein-binding and membrane-fusion events. After transport, GTPase-activating
proteins (GAPS) in
the target membrane are responsible for converting the GTP-bound Rab proteins
to their GDP-bound
state. Finally, guanine-nucleotide dissociation inhibitor (GDI) recruiter the
GDP-bound proteins to
their membrane of origin.
Other regulators of G-protein signaling (RGS) also exist that act primarily by
negatively
regulating the G-protein pathway by an unknown mechanism (Druey, K.M. et al. (
1996) Nature
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379:742-746). Some 15 members of the RGS family have been identified. RGS
family members are
related structurally through similarities in an approximately 120 amino acid
region termed the RGS
domain and functionally by their ability to inhibit the interleukin (cytokine)
induction of MAP kinase in
cultured mammalian 293T cells (Druey et al., su ra).
A member of the Rho family of G-proteins is CDC42, a regulator of cytoskeletal
rearrangements required for cell division. CDC42 is inactivated by a specific
GAP (CDC42GAP) that
strongly stimulates the GTPase activity of CDC42 while having a much lesser
effect on other Rho
family members. CDC42GAP also contains an SH3 binding domain that interacts
with the SH3
domains of cell signaling proteins such as p85 alpha and c-Src, suggesting
that CDC42GAP may serve
as a link between CDC42 and other cell signaling pathways (Barfod, E.T. et al.
(1993) J. Biol. Chem.
268:26059-26062).
The Dbl proteins are a family of GEFs for the Rho and Ras G-proteins
(Whitehead, LP. et al.
(1997) Biochim. Biophys. Acta 1332:F1-F23). All Dbl family members contain a
Dbl homology (DH)
domain of approximately 180 amino acids, as well as a pleckstrin homology (PH)
domain located
immediately C-terminal to the DH domain. Most Dbl proteins have oncogenic
activity, as
demonstrated by the ability to transform various cell lines, consistent with
roles as regulators of Rho-
mediated oncogenic signaling pathways. The kalirin proteins are neuron-
specific members of the Dbl
family, which are located to distinct subcellular regions of cultured neurons
(Johnson, R.C. (2000) J.
Cell Biol. 275:19324-19333).
Other regulators of G-protein signaling (RGS) also exist that act primarily by
negatively
regulating the G-protein pathway by an unknown mechanism (Druey, K.M. et al.
(1996) Nature
379:742-746). Some 15 members of the RGS family have been identified. RGS
family members are
related structurally through similarities in an approximately 120 amino acid
region termed the RGS
domain and functionally by their ability to inhibit the interleukin (cytokine)
induction of MAP kinase in
cultured mammalian 293T cells (Druey et al., supy~a).
The Tm_m__uno-associated nucleotide (IAN) family of proteins has GTP-binding
activity as
indicated by the conserved ATP/GTP-binding site P-loop motif. The IAN family
includes IAN-1,
IAN-4, IAP38, and IAG-1. IAN-1 is expressed in the immune system, specifically
in T cells and
thyrriocytes. Its expression is induced during thymic events (Poirier, G.M.C.
et al. (1999) J. Tmm__unol.
163:4960-4969). IAP38 is expressed in B cells and macrophages and its
expression is induced in
splenocytes by pathogens. IAG-1, which is a plant molecule, is induced upon
bacterial infection
(Krucken, J. et al. (1997) Biochem. Biophys. Res. Commun. 230:167-170). IAN-4
is a mitochondrial
membrane protein which is preferentially expressed in hematopoietic precursor
32D cells transfected
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with wild-type versus mutant forms of the bcr/abl oncogene. The bcr/abl
oncogene is known to be
associated with chronic myelogenous leukemia, a clonal myelo-proliferative
disorder, which is due to
the translocation between the bcr gene on chromosome 22 and the abl gene on
chromosome 9. Bcr is
the breakpoint cluster region gene and abl is the cellular homolog of the
transforming gene of the
Abelson murine leukemia virus. Therefore, the IAN family of proteins appears
to play a role in cell
survival in immune responses and cellular transformation (Daheron, L. et al.
(2001) Nucleic Acids
Res. 29:1308-1316).
Formin-related genes (FRL) comprise a large family of morphoregulatory genes
and have
been shown to play important roles in morphogenesis, embryogenesis, cell
polarity, cell migration, and
cytokinesis through their interaction with Rho family small GTPases. Formin
was first identified in
mouse limb defofmity (lel) mutants where the distal bones and digits of all
limbs are fused and
reduced in size. FRL contains formin homology domains FH1, FH2, and FH3. The
FH1 domain has
been shown to bind the Src homology 3 (SH3) domain, WWP/WW domains, and
profilin. The FHZ
domain is conserved and was shown to be essential for formin function as
disruption at the FHZ
domain results in the characteristic ld phenotype. The FH3 domain is located
at the N-terminus of
FRL, and is required for associating with Rac, a Rho family GTPase (Yayoshi-
Yamamoto, S. et al.
(2000) Mol. Cell. Biol. 20:6872-6881).
Signaling Complex Protein Domains
PDZ domains were named for three proteins in, which this domain was initially
discovered.
2o These proteins include PSD-95 (postsynaptic density 95), Dlg (Df-osophila
lethal(1)discs large-1), and
ZO-1 (zonula occludens-1). These proteins play important roles in neuronal
synaptic transmission,
tumor suppression, and cell junction formation, respectively. Since the
discovery of these proteins,
over sixty additional PDZ-containing proteins have been identified in diverse
prokaryotic and
eukaryotic organisms. This domain has been implicated in receptor and ion
channel clustering and in
the targeting of multiprotein signaling complexes to specialized functional
regions of the cytosolic face
of the plasma membrane. (For a review of PDZ domain-containing proteins, see
Pouting, C.P. et al.
(1997) Bioessays 19:469-479.) A large proportion of PDZ domains are found in
the eukaryotic
MAGUK (membrane-associated guanylate kinase) protein family, members of which
bind to the
intracellular domains of receptors and channels. However, PDZ domains are also
found in diverse
membrane-localized proteins such as protein tyrosine phosphatases,
serine/threonine kinases, G-protein
cofactors, and synapse-associated proteins such as syntrophins and neuronal
nitric oxide synthase
(nNOS). Generally, about one to three PDZ domains are found in a given
protein, although up to nine
PDZ domains have been identified in a single protein. The glutamate receptor
interacting protein
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(GRIP) contains seven PDZ domains. GRIP is an adaptor that links certain
glutamate receptors to
other proteins and may be responsible for the clustering of these receptors at
excitatory synapses in
the brain (Dong, H. et al. (1997) Nature 386:279-284). The Drosophila scribble
(SCRIB) protein
contains both multiple PDZ domains and leucine-rich repeats. SCRIB is located
at the epithelial
septate junction, which is analogous to the vertebrate tight junction, at the
boundary of the apical and
basolateral cell surface. SCRIB is involved in the distribution of apical
proteins and correct placement
of adherens junctions to the basolateral cell surface (Bilder, D. and N.
Perrimon (2000) Nature
403:676-680).
The PX domain is an example of a domain specialized for promoting protein-
protein
interactions. The PX domain is found in sorting nexins and in a variety of
other proteins, including the
PhoX components of NADPH oxidase and the Cpk class of phosphatidylinositol 3-
kinase. Most PX
domains contain a polyproline motif which is characteristic of SH3 domain-
binding proteins (Pouting,
C.P. (1996) Protein Sci. 5:2353-2357). SH3 domain-mediated interactions
involving the PhoX
components of NADPH oxidase play a role in the formation of the NADPH oxidase
multi-protein
complex (Leto, T.L. et al. (1994) Proc. Natl. Acad. Sci. USA 91:10650-10654;
Wilson, L. et al.
(1997) Inflamm. Res. 46:265-271).
The SH3 domain is defined by homology to a region of the proto-oncogene c-Src,
a
cytoplasmic protein tyrosine kinase. SH3 is a small domain of 50 to 60 amind
acids that interacts with
proline-rich ligands. SH3 domains are found in a variety of eukaryotic
proteins involved in signal
transduction, cell polarization, and membrane-cytoskeleton interactions. In
some cases, SH3 domain-
containing proteins interact directly with receptor tyrosine kinases. For
example, the SLAP-130
protein is a substrate of the T-cell receptor (TCR) stimulated protein kinase.
SLAP-130 interacts via
its SH3 domain with the protein SLP-76 to affect the TCR-induced expression of
interleukin-2 (Musci,
M.A. et al. (1997) J. Biol. Chem. 272:11674-11677). Another recently
identified SH3 domain protein
is macrophage actin-associated tyrosine-phosphorylated protein (MAYP) which is
phosphorylated
during the response of macrophages to colony stimulating factor-1 (CSF-1) and
is likely to play a role
in regulating the CSF-1-induced reorganization of the actin cytoskeleton
(Yeung, Y.-G. et al. (1998) J.
Biol. Chem. 273:30638-30642). The structure of the SH3 domain is characterized
by two antiparallel
beta sheets packed against each other at right angles. This packing forms a
hydrophobic pocket lined
with residues that are highly conserved between different SH3 domains. This
pocket makes critical
hydrophobic contacts with proline residues in the ligand (Feng, S. et al.
(1994) Science 266:1241-
1247).
A novel domain, called the WW domain, resembles the SH3 domain in its ability
to bind
CA 02458645 2004-02-16
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proline-rich ligands. This domain. was originally discovered in dystrophin, a
cytoskeletal protein with
direct involvement in Duchenne muscular dystrophy (Bork, P. and M. Sudol
(1994) Trends Biochem.
Sci. 19:531-533). WW domains have since been discovered in a variety of
intracellular signaling
molecules involved in development, cell differentiation, and cell
proliferation. The structure of the
WW domain is composed of beta strands grouped around four conserved aromatic
residues, generally
tryptophan.
Like SH3, the SH2 domain is defined by homology to a region of c-Src. SHZ
domains interact
directly with phospho-tyrosine residues, thus providing an immediate mechanism
for the regulation and
transduction of receptor tyrosine kinase-mediated signaling pathways. For
example, as many as ten
distinct SH2 domains are capable of binding to phosphorylated tyrosine
residues in the activated PDGF
receptor, thereby providing a highly coordinated and finely tuned response to
ligand-mediated receptor
activation. (Reviewed in Schaffhausen, B. (1995) Biochim. Biophys. Acta.
1242:61-7S.) The BLNK
protein is a linker protein involved in B cell activation, that bridges B cell
receptor-associated kinases
with SH2 domain effectors that link to various signaling pathways (Fu, C. et
al. (1998) Immunity 9:93-
1S 103).
The pleckstrin homology (PH) domain was originally identified in pleckstrin,
the predominant
substrate for protein kinase C in platelets. Since its discovery, this domain
has been identified in over
90 proteins involved in intracellular signaling or cytoskeletal organization.
Proteins containing the
pleckstrin homology domain include a variety of kinases, phospholipase-C
isoforms, guanine nucleotide
release factors, and GTPase activating proteins. For example, members of the
FGD1 family contain
both Rho-guanine nucleotide exchange factor (GEF) and PH domains, as well as a
FYVE zinc finger
domain. FGD1 is the gene responsible for faciogenital dysplasia, an inherited
skeletal dysplasia
(Pasteris, N.G. and J.L. Gorski (1999) Genomics 60:57-66). Many PH domain
proteins function in
association with the plasma membrane, and this association appears to be
mediated by the PH domain
2S itself. PH domains share a common structure composed of two antiparallel
beta sheets flanked by an
amplupathic alpha helix. Variable loops connecting the component beta strands
generally occur within
a positively charged environment and may function as ligand binding sites
(Lemmon, M.A. et al.
(1996) Cell 85:621-624). Ankyrin (ANK) repeats mediate protein-protein
interactions
associated with diverse intracellular signaling functions. For example, ANK
repeats are found in
3o proteins involved in cell proliferation such as kinases, kinase inhibitors,
tumor suppressors, and cell
cycle control proteins. (See, for example, Kalus, W. et al. (1997) FEBS Lett.
401:127-132; Ferrante,
A.W. et al. (1995) Proc. Natl. Acad. Sci. USA 92:1911-1915.) These proteins
generally contain
multiple ANK repeats, each composed of about 33 amino acids. Myotrophin is an
ANK repeat
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protein that plays a key role in the development of cardiac hypertrophy, a
contributing factor to many
heart diseases. Structural studies show that the myotrophin ANK repeats, like
other ANK repeats,
each form a helix-turn-helix core preceded by a protruding "tip." These tips
are of variable sequence
and may play a role in protein-protein interactions. The helix-turn-helix
region of the ANK repeats
stack on top of one another and are stabilized by hydrophobic interactions
(Yang, Y. et al. (1998)
Structure 6:619-626). Members of the ASB protein family contain a suppressor
of cytokine signaling
(SOCS) domain as well as multiple ankyrin repeats (Hilton, D.J. et al. (1998)
Proc. Natl. Acad. Sci.
USA 95:114-119).
The tetratricopeptide repeat (T'PR) is a 34 amino acid repeated motif found in
organisms from
bacteria to humans. T'PRs are predicted to form ampipathic helices, and appear
to mediate protein-
protein interactions. T'PR domains are found in CDC16, CDC23, and CDC27,
members of the
anaphase promoting complex which targets proteins for degradation at the onset
of anaphase. Other
processes involving T'PR proteins include cell cycle control, transcription
repression, stress response,
and protein kinase inhibition (Lamb, J.R. et al. (1995) Trends Biochem. Sci.
20:257-259).
The armadillo/beta-catenin repeat is a 42 amino acid motif which forms a
superhelix of alpha
helices when tandemly repeated. The structure of the armadillo repeat region
from beta-catenin
revealed a shallow groove of positive charge on one face of the superhelix,
which is a potential binding
surface. The armadillo repeats of beta-catenin, plakoglobin, and p120°~
bind the cytoplasmic domains
of cadherins. Beta-catenin/cadherin complexes are targets of regulatory
signals that govern cell
adhesion and mobility (Huber, A.H. et al. (1997) Cell 90:871-882).
Eight tandem repeats of about 40 residues (WD-40 repeats), each containing a
central
Trp-Asp motif, make up beta-transducin (G-beta), which is one of the three
subunits (alpha, beta, and
gamma) of the guanine nucleotide-binding proteins (G proteins). In higher
eukaryotes G-beta exists as
a small multigene family of highly conserved proteins of about 340 amino acid
residues. WD repeats
are also found in other protein families. For example, betaTRCP is a component
of the ubiquitin ligase
complex, which recruits specific proteins, including beta-catenin, to the
ubiquitin-proteasome
degradation pathway. BetaTRCP and its isoforms all contain seven WD repeats,
as well as a
characteristic "F box" motif. (Koike, J. et al. (2000) Biochem. Biophys. Res.
Commun. 269:103-109.)
Signaling by Notch family receptors controls cell fate decisions during
development (Frisen, J.
and Lendahl, U. (2001) Bioessays 23:3-7). The Notch receptor signaling pathway
is involved in the
morphogenesis and development of many organs and tissues in multicellular
species. Notch receptors
are large transmembrane proteins that contain extracellular regions made up of
repeated EGF
domains. Notchless was identified in a screen for molecules that modulate
notch activity (Royet, J. et
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al. (1998) EMBO J. 17:7351-7360). Notchless, which contains nine WD40 repeats,
binds to the
cytoplasmic domain of Notch and inhibits Notch activity. Eps8 is a substrate
for the intracellular
epidermal growth factor receptors (EGFR).
Semaphorins are secreted, glycosylphosphatidylinositol (GPI) anchor and
transmembrane
glycoproteins. Semaphorins function as chemorepellants in various sensory and
motor axons (Soker,
S. (2001) Int. J. Biochem. Cell Biol. 33:433-437). Semaphorins constitute one
type of ligand for the
plexin receptor.
Tumor necrosis factor receptor-associated factors (TRAFs) constitute a family
of adaptor
proteins that link the cytosolic domains of these receptors to downstream
protein kinases or WD
repeats are also found in other protein families. For example, betaTRCP is a
component of the
ubiquitin ligases. These proteins share a TRAF domain (TD), a distinctive
region near the COOH
terminus, that is responsible for mediating interactions between TRAFs and TNF
receptors with other
adaptor proteins and kinases.
Expression profiling
Microarrays are analytical tools used in bioanalysis. A microarray has a
plurality of molecules
spatially distributed over, and stably associated with, the surface of a solid
support. Microarrays of
polypeptides, polynucleotides, and/or antibodies have been developed and find
use in a variety of
applications, such as gene sequencing, monitoring gene expression, gene
mapping, bacterial
identification, drug discovery, and combinatorial chemistry.
One area in particular in which microarrays find use is in gene expression
analysis. Array
technology can provide a simple way to explore the expression of a single
polymorphic gene or the
expression profile of a large number of related or unrelated genes. When the
expression of a single
gene is examined, arrays are employed to detect the expression of a specific
gene or its variants.
When an expression profile is examined, arrays provide a platform for
identifying genes that are tissue
specific, are affected by a substance being tested in a toxicology assay, are
part of a signaling
cascade, carry out housekeeping functions, or are specifically related to a
particular genetic
predisposition, condition, disease, or disorder.
Steroid hormones
Steroids are a class of lipid-soluble molecules, including cholesterol, bile
acids, vitamin D, and
hormones, that share a common four-ring structure based on
cyclopentanoperhydrophenanthrene and
that carrry out a wide variety of functions. Cholesterol, for example, is a
component of cell
membranes that controls membrane fluidity. It is also a precursor for bile
acids which solubilize lipids
and facilitate absorption in the small intestine during digestion. Vitamin D
regulates the absorption of
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calcium in the small intestine and controls the concentration of calcium in
plasma. Steroid hormones,
produced by the adrenal cortex, ovaries, and testes, include glucocorticoids,
mineralocorticoids,
androgens, and estrogens. They control various biological processes by binding
to intracellular
receptors that regulate transcription of speci~.c genes in the nucleus.
Glucocorticoids, for example,
increase blood glucose concentrations by regulation of gluconeogenesis in the
liver, increase blood
concentrations of fatty acids by promoting lipolysis in adipose tissues,
modulate sensitivity to
catcholaxnines in the central nervous system, and reduce inflammation. The
principal
mineralocorticoid, aldosterone, is produced by the adrenal cortex and acts on
cells of the distal tubules
of the kidney to enhance sodium ion reabsorption. Androgens, produced by the
interstitial cells of
Leydig in the testis, include the male sex hormone testosterone, which
triggers changes at puberty, the
production of sperm and maintenance of secondary sexual characteristics.
Female sex hormones,
estrogen and progesterone, are produced by the ovaries and also by the
placenta and adrenal cortex of
the fetus during pregnancy. Estrogen regulates female reproductive processes
and secondary sexual
characteristics. Progesterone regulates changes in the endometrium during the
menstrual cycle and
pregnancy.
Steroid hormones are widely used for fertility control and in anti-
inflammatory treatments for
physical injuries and diseases such as arthritis, asthma, and auto-immune
disorders. Progesterone, a
naturally occurring progestin, is primarily used to treat amenorrhea, abnormal
uterine bleeding, or as a
contraceptive. Endogenous progesterone is responsible for inducing secretory
activity in the
endometrium of the estrogen-primed uterus in preparation for the implantation
of a fertilized egg and
for the maintenance of pregnancy. It is secreted from the corpus luteum in
response to luteinizing
hormone (LH). The primary contraceptive effect of exogenous progestins
involves the suppression
of the midcycle surge of LH. At the cellular level, progestins diffuse freely
into target cells and bind
to the progesterone receptor. Target cells include the female reproductive
tract, the mammary gland,
the hypothalamus, and the pituitary. Once bound to the receptor, progestins
slow the frequency of
release of gonadotropin releasing hormone from the hypothalamus and blunt the
pre-ovulatory LH
surge, thereby preventing follicular maturation and ovulation. Progesterone
has ini"mal estrogenic
and androgenic activity. Progesterone is metabolized hepatically to
pregnanediol and conjugated with
glucuronic acid.
3o Medroxyprogesterone (MAH), also known as hoc-methyl-17-hydroxyprogesterone,
is a
synthetic progestin with a pharmacological activity about 15 times greater
than progesterone. MAH is
used for the treatment of renal and endometrial carcinomas, amenorrhea,
abnormal uterine bleeding,
and endometriosis associated with hormonal imbalance. MAH has a stimulatory
effect on respiratory
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centers and has been used in cases of low blood oxygenation caused by sleep
apnea, chronic
obstructive pulmonary disease, or hypercapnia.
Mifepristone, also known as RU-486, is an antiprogesterone drug that blocks
receptors of
progesterone. It counteracts the effects of progesterone, which is needed to
sustain pregnancy.
Mifepristone induces spontaneous abortion when administered in early pregnancy
followed by
treatment with the prostaglandin, misoprostol. Further, studies show that
mifepristone at a
substantially lower dose can be highly effective as a postcoital contraceptive
when administered within
five days after unprotected intercourse, thus providing women with a "morning-
after pill" in case of
contraceptive failure or sexual assault. Mifepristone also has potential uses
in the treatment of breast
and ovarian cancers in cases in which tumors are progesterone-dependent. It
interferes with steroid-
dependent growth of brain meningiomas, and may be useful in treatment of
endometriosis where it
blocks the estrogen-dependent growth of endometrial tissues. It may also be
useful in treatment of
uterine fibroid tumors and Cushing's Syndrome. Mifepristone binds to
glucocorticoid receptors and
interferes with cortisol binding. Mifepristone also may act as an anti-
glucocorticoid and be effective
for treating conditions where cortisol levels are elevated such as AIDS,
anorexia nervosa, ulcers,
diabetes, Parkinson's disease, multiple sclerosis, and Alzheimer's disease.
Danazol is a synthetic steroid derived from ethinyl testosterone. Danazol
indirectly reduces
estrogen production by lowering pituitary synthesis of follicle-stimulating
hornione and LH. Danazol
also binds to sex hormone receptors in target tissues, thereby exhibiting
anabolic, antiestrognic, and
weakly androgenic activity. Danazol does not possess any progestogenic
activity, and does not
suppress normal pituitary release of corticotropin or release of cortisol by
the adrenal glands. Danazol
is used in the treatment of endometriosis to relieve pain and inhibit
endometrial cell growth. It is also
used to treat fibrocystic breast disease and hereditary angioedema.
Corticosteroids are used to relieve inflammation and to suppress the immune
response. They
inhibit eosinophil, basophil, and airway epithelial cell function by
regulation of cytokines that mediate
the inflammatory response. They inhibit leukocyte infiltration at the site of
inflammation, interfere in.
the function of mediators of the inflammatory response, and suppress the
humoral immune~response.
Corticosteroids are used to treat allergies, asthma, arthritis, and skin
conditions. Beclomethasone is a
synthetic glucocorticoid that is used to treat steroid-dependent asthma, to
relieve symptoms associated
with allergic or nonallergic (vasomotor) rhinitis, or to prevent recurrent
nasal polyps following surgical
removal. The anti-inflammatory and vasoconstrictive effects of intranasal
beclomethasone are 5000
times greater than those produced by hydrocortisone. Budesonide is a
corticosteroid used to control
symptoms associated with allergic rhinitis or asthma. . Budesonide has high
topical anti-inBammatory
CA 02458645 2004-02-16
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activity but low systemic activity. Dexamethasone is a synthetic
glucocorticoid used in anti-
inflammatory or immunosuppressive compositions. It is also used in inhalants
to prevent symptoms of
asthma. Due to its greater ability to reach the central nervous system,
dexamethasone is usually the
treatment of choice to control cerebral edema. Dexamethasone is approximately
20-30 times more
potent than hydrocortisone and 5-7 times more potent than prednisone.
Prednisone is metabolized in
the liver to its active form, prednisolone, a glucocorticoid with anti-
inflammatory properties.
Prednisone is approximately 4 times more potent than hydrocortisone and the
duration of action of
prednisone is intermediate between hydrocortisone and dexamethasone.
Prednisone is used to treat
allograft rejection, asthma, systemic lupus erythematosus, arthritis,
ulcerative colitis, and other
inflammatory conditions. Betamethasone is a synthetic glucocorticoid with
antiinflammatory and
immunosuppressive activity and is used to treat psoriasis and fungal
infections, such as athlete's foot
and ringworm.
The anti-inflammatory actions of corticosteroids are thought to involve
phospholipase Aa
inhibitory proteins, collectively called lipocortins. Lipocortins, in turn,
control the biosynthesis of potent
mediators of inflammation such as prostaglandins and leukotrienes by
inhibiting the release of the
precursor molecule arachidonic acid. Proposed mechanisms of action include
decreased IgE
synthesis, increased number of (3-adrenergic receptors on leukocytes, and
decreased arachidonic acid
metabolism. During an immediate allergic reaction, such as in chronic
bronchial asthma, allergens
bridge the IgE antibodies on the surface of mast cells, which triggers these
cells to release
chemotactic substances. Mast cell influx and activation, therefore, is
partially responsible for the
inflammation and hyperirritability of the oral mucosa in asthmatic patients.
This inflammation can be
retarded by administration of corticosteroids.
Tmmune response cells and proteins
Human peripheral blood mononuclear cells (PBMCs) contain B lymphocytes, T
lymphocytes,
NK cells, monocytes, dendritic cells and progenitor cells.
Glucocorticoids are naturally occurring hormones that prevent or suppress
inflammation and
immune responses when administered at pharmacological doses. Unbound
glucocorticoids readily
cross cell membranes and bind with high affinity to specific cytoplasmic
receptors. Subsequent to
binding, transcription and protein synthesis are affected. The result can
include inhibition of leukocyte
infiltration at the site of inflammation, interference in the function of
mediators of inflammatory
response, and suppression of humoral immune responses. The anti-inflammatory
actions of
corticosteroids are thought to involve phospholipase A2 inhibitory proteins,
collectively called
lipocortins. Lipocortins, in turn, control the biosynthesis of potent
mediators of inflammation such as
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prostaglandins and leukotrienes by inhibiting the release of the precursor
arachidonic molecule.
Staphylococcal exotoxins specifically activate human T cells, expressing an
appropriate TCR-
Vbeta chain. Although polyclonal in nature, T cells activated by
Staphylococcal exotoxins require
antigen presenting cells (APCs) to present the exotoxin molecules to the T
cells and deliver the
costimulatory signals required for optimum T cell activation. Although
Staphylococcal exotoxins must
be presented to T cells by APCs, these molecules need not be processed by APC.
Staphylococcal
exotoxins directly bind to a non-polymorphic portion of the human MHC class 1I
molecules, bypassing
the need for capture, cleavage, and binding of the peptides to the polymorphic
antigenic groove of the
MHC class II molecules.
Colon cancer
The potential application of gene expression profiling is particularly
relevant to improving
diagnosis, prognosis, and treatment of cancers, such as colon cancer. Colon
cancer evolves through a
multi-step process whereby pre-malignant colonocytes undergo a relatively
defined sequence of
events leading to tumor formation. Several factors participate in the process
of tumor progression and
malignant transformation including genetic factors, mutations, and selection.
To understand the nature of gene alterations in colorectal cancer, a number of
studies have
focused on the inherited syndromes. Familial adenomatous polyposis (FAP), is
caused by mutations in
the adenomatous polyposis coli gene (APC), resulting in truncated or inactive
forms of the protein.
This tumor suppressor gene has been mapped to chromosome Sq. Hereditary
nonpolyposis colorectal
cancer (HNPCC) is caused by mutations in mis-match repair genes. Although
hereditary colon
cancer syndromes occur in a small percentage of the population and most
colorectal cancers are
considered sporadic, knowledge from studies of the hereditary syndromes can be
generally applied.
For instance, somatic mutations in APC occur in at least 80% of sporadic colon
tumors. APC
mutations are thought to be the initiating event in the disease. Other
mutations occur subsequently.
Approximately 50% of colorectal cancers contain activating mutations in ras,
while 85% contain
inactivating mutations in p53. Changes in all of these genes lead to gene
expression changes in colon
cancer.
There is a need in the art for new compositions, including nucleic acids and
proteins, for the
diagnosis, prevention, and treatment of cell proliferative, endocrine,
autoimmune/inflammatory,
neurological, gastrointestinal, reproductive, developmental, and vesicle
trafficking disorders.
SUMMARY OF THE INVENTION
Various embodiments of the invention provide purified polypeptides,
intracellular signaling
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molecules, referred to collectively as 'INTSIG' and individually as 'INTSIG-
1,' 'INTSIG-2,'
'INTSIG-3,' 'JNTSIG-4,' '1NTSIG-5,' '1NTSIG-6,' 'INTSIG-7,' 'INTSIG-8,'
'1NTSIG-9,' '1NTSIG-
10,' 'INTSIG-11,' 'INTSIG-12,' 'INTSIG-13,' '1NTSIG-14,' '7NTSIG-15,' '1NTSIG-
16,' 'INTSIG-
17,' '1NTSIG-18,' '1NTSIG-19,' 'INTSIG-20,' '1NTSIG-21,' '1NTSIG-22,' 'INTSIG-
23,' 'INTSIG-
24,' 'INTSIG-25,' 'lN'TSIG-26,' 'INTSIG-27,' 'INTSIG-28,' 'INTSIG-29,' 'INTSIG-
30,' 'INTSIG-
31,' 'INTSIG-32,' 'INTSIG-33,' 'INTSIG-34,' 'INTSIG-35,' 'INTSIG-36,'
'IN'I'SIG-37,' 'INTSIG-
38,' '1NTSIG-39,' 'INTSIG-40,' 'INTSIG-41,' 'INTSIG-42,' 'JNTSIG-43,' '1NTSIG-
44,' and
'INTSIG-45' and methods for using these proteins and their encoding
polynucleotides for the detection,
diagnosis, and treatment of diseases and medical conditions. Embodiments also
provide methods for
utilizing the purified intracellular signaling molecules and/or their encoding
polynucleotides for
facilitating the drug discovery process, including determination of efficacy,
dosage, toxicity, and
pharmacology. Related embodiments provide methods for utilizing the purified
intracellular signaling
molecules and/or their encoding polynucleotides for investigating the
pathogenesis of diseases and
medical conditions.
An embodiment provides an isolated polypeptide selected from the group
consisting of a) a
polypeptide comprising an amino acid sequence selected from the group
consisting of SEQ ID N0:1-
45, b) a polypeptide comprising a naturally occurring amino acid sequence at
least 90% identical or at
least about 90% identical to an amino acid sequence selected from the group
consisting of SEQ ll~
N0:1-45, c) a biologically active fragment of a polypeptide having an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-45, and d) an immunogenic fragment of
a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-45. Another
embodiment provides an isolated polypeptide comprising an amino acid sequence
of SEQ ll~ N0:1-45.
Still another embodiment provides an isolated polynucleotide encoding a
polypeptide selected
from the group consisting of a) a polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ ID N0:1-45, b) a polypeptide comprising a naturally
occurring amino acid
sequence at least 90% identical or at least about 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ~ N0:1-45, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-45, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-45. In another embodiment, the polynucleotide
encodes a polypeptide
selected from the group consisting of SEQ ID N0:1-45. In an alternative
embodiment, the
polynucleotide is selected from the group consisting of SEQ )D NO:46-90.
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Still another embodiment provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ )D NO:1-45, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical or at least about 90% identical to an amino acid sequence
selected from the group
consisting of SEQ ll~ N0:1-45, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ >D N0:1-45, and d) an
immunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ m N0:1-45.
Another embodiment provides a cell transformed with the recombinant
polynucleotide. Yet another
embodiment provides a transgenic organism comprising the recombinant
polynucleotide.
Another embodiment provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ll~ N0:1-45, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical or at least about 90% identical to an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-45, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ ll~ N0:1-45, and d) an
immunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ll~ NO:1-45.
The method comprises a) culturing a cell under conditions suitable for
expression of the polypeptide,
wherein said cell is transformed with a recombinant polynucleotide comprising
a promoter sequence
operably linked to a polynucleotide encoding the polypeptide, and b)
recovering the polypeptide so
expressed.
Yet another embodiment provides an isolated antibody which specifically binds
to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid sequence
selected from the group consisting of SEQ )D N0:1-45, b) a polypeptide
comprising a naturally
occurring amino acid sequence at least 90%o identical or at least about 90%
identical to an amino acid
sequence selected from the group consisting of SEQ ID N0:1-45, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ff7 N0:1-45,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID N0:1-45.
Still yet another embodiment provides an isolated polynucleotide selected from
the group
consisting of a) a polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:46-90, b) a polynucleotide comprising a naturally
occurring polynucleotide
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sequence at least 90% identical or at least about 90% identical to a
polynucleotide sequence selected
from the group consisting of SEQ )D N0:46-90, c) a polynucleotide
complementary to the
polynucleotide of a), d) a polynucleotide complementary to the polynucleotide
of b), and e) an RNA
equivalent of a)-d). In other embodiments, the polynucleotide can comprise at
least about 20, 30, 40,
60, 80, or 100 contiguous nucleotides.
Yet another embodiment provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide being selected from the group consisting of a) a
polynucleotide comprising a
polynucleotide sequence selected from the group consisting of SEQ ll~ N0:46-
90, b) a polynucleotide
comprising a naturally occurring polynucleotide sequence at least 90%
identical or at least about 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID NO:46-90, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises
a) hybridizing the
sample with a probe comprising at least 20 contiguous nucleotides comprising a
sequence
complementary to said target polynucleotide in the sample, and which probe
specifically hybridizes to
said target polynucleotide, under conditions whereby a hybridization complex
is formed between said
probe and said target polynucleotide or fragments thereof, and b) detecting
the presence or absence of
said hybridization complex. In a related embodiment, the method can include
detecting the amount of
the hybridization complex. In still other embodiments, the probe can comprise
at least about 20, 30,
40, 60, 80, or 100 contiguous nucleotides.
Still yet another embodiment provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide being selected from the group consisting of
a) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:46-90, b) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical or at
least about 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ ll~
N0:46-90, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method
comprises a) amplifying said target polynucleotide or fragment thereof using
polymerase chain
reaction amplification, and b) detecting the presence or absence of said
amplified target polynucleotide
or fragment thereof. In a related embodiment, the method can include detecting
the amount of the
amplified target polynucleotide or fragment thereof.
Another embodiment provides a composition comprising an effective amount of a
polypeptide
selected from the group consisting of a) a polypeptide comprising an. amino
acid sequence selected
CA 02458645 2004-02-16
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from the group consisting of SEQ 117 N0:1-45, b) a polypeptide comprising a
naturally occurring
ammo acid sequence at least 90% identical or at least about 90% identical to
an amino acid sequence
selected from the group consisting of SEQ ID N0:1-45, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ 1D N0:1-45,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ 1D NO:1-45, and a pharmaceutically acceptable
excipient. In one
embodiment, the composition can comprise an amino acid sequence selected from
the group consisting
of SEQ ll~ N0:1-45. Other embodiments provide a method of treating a disease
or condition
associated with decreased or abnormal expression of functional INTSIG,
comprising administering to a
patient in need of such treatment the composition.
Yet another embodiment provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID N0:1-45, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical or at least
about 90% identical to an
amino acid sequence selected from the group consisting of SEQ ll~ NO:1-45, c)
a biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID N0:1-45, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ ll~ N0:1-45. The method comprises a)
exposing a sample
comprising the polypeptide to a compound, and b) detecting agonist activity in
the sample. Another
embodiment provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. Yet another embodiment provides a
method of treating a
disease or condition associated with decreased expression of functional
1NTSIG, comprising
administering to a patient in need of such treatment the composition.
Still yet another embodiment provides a method for screening a compound for
effectiveness
as an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID N0:1-45, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical or
at least about 90%
identical to an amino acid sequence selected from the group consisting of SEQ
ID NO:1-45, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-45, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ~ N0:1-45. The method
comprises a)
exposing a sample comprising the polypeptide to a compound, and b) detecting
antagonist activity in
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CA 02458645 2004-02-16
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the sample. Another embodiment provides a composition comprising an antagonist
compound
identified by the method and a pharmaceutically acceptable excipient. Yet
another embodiment
provides a method of treating a disease or condition associated with
overexpression of functional
1NTSIG, comprising administering to a patient in need of such treatment the
composition.
Another embodiment provides a method of screening for a compound that
specifically binds to
a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID N0:1-45, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical or at least
about 90% identical to an
amino acid sequence selected from the group consisting of SEQ ID N0:1-45, c) a
biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID N0:1-45, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ ll~ NO:1-45. The method comprises a)
combining the
polypeptide with at least one test compound under suitable conditions, and b)
detecting binding of the
polypeptide to the test compound, thereby identifying a compound that
specifically binds to the
polypeptide.
Yet another embodiment provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID N0:1-45, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical or at least
about 90% identical to an
amino acid sequence selected from the group consisting of SEQ ~ N0:1-45, c) a
biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID N0:1-45, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-45. The method comprises a)
combining the
polypeptide with at least one test compound under conditions permissive for
the activity of the
polypeptide, b) assessing the activity of the polypeptide in the presence of
the test compound, and c)
comparing the activity of the polypeptide in the presence of the test compound
with the activity of the
polypeptide in the absence of the test compound, wherein a change in the
activity of the polypeptide in
the presence of the test compound is indicative of a compound that modulates
the activity of the
polypeptide.
Still yet another embodiment provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
polynucleotide sequence selected from the group consisting of SEQ ll~ NO:46-
90, the method
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comprising a) exposing a sample comprising the target polynucleotide to a
compound, b) detecting
altered expression of the target polynucleotide, and c) comparing the
expression of the target
polynucleotide in the presence of varying amounts of the compound and in the
absence of the
compound.
Another embodiment provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound; b)
hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
m NO:46-90, ii) a
1o polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical or at
least about 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:46-90, iii) a polynucleotide having a sequence complementary to i), iv) a
polynucleotide
complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-
iv). Hybridization occurs
under conditions whereby a specific hybridization complex is formed between
said probe and a target
15 polynucleotide in the biological sample, said target polynucleotide
selected from the group consisting of
i) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
ID N0:46-90, ii) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical or at least about 90% identical to a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:46-90, iii) a polynucleotide complementary to the
polynucleotide of i), iv) a
20 polynucleotide complementary to the polynucleotide of ii), and v) an RNA
equivalent of i)-iv).
Alternatively, the target polynucleotide can comprise a fragment of a
polynucleotide selected from the
group consisting of i)-v) above; c) quantifying the amount of hybridization
complex; and d) comparing
the amount of hybridization complex in the treated biological sample with the
amount of hybridization
complex in an untreated biological sample, wherein a difference in the amount
of hybridization
25 complex in the treated biological sample is indicative of toxicity of the
test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for full length polynucleotide and
polypeptide
embodiments of the invention.
30 Table 2 shows the GenBank identification number and annotation of the
nearest GenBank
homolog, and the PROTEOME database identification numbers and annotations of
PROTEOME
database homologs, for polypeptide embodiments of the invention. The
probability scores for the
2s
CA 02458645 2004-02-16
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matches between each polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide embodiments, including
predicted motifs and
domains, along with the methods, algorithms, and searchable databases used for
analysis of the
polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide embodiments, along with selected fragments of the
polynucleotides.
Table S shows representative cDNA libraries for polynucleotide embodiments.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze
polynucleotides and
polypeptides, along with applicable descriptions, references, and threshold
parameters.
Table 8 .shows single nucleotide polymorphisms found in polynucleotide
sequences of the
invention, along with allele frequencies in different human populations.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleic acids, and methods are described, it is
understood that
embodiments of the invention are not limited to the particular machines,
instruments, materials, and
methods described, as these may vary. It is also to be understood that the
terminology used herein is
for the purpose of describing particular embodiments only, and is not intended
to limit the scope of the
invention.
As used herein and in the appended claims, the singular forms "a," "an," and
"the" include
plural reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a
host cell" includes a plurality of such host cells, and a reference to "an
antibody" is a reference to one
or more antibodies and equivalents thereof known to those skilled in the art,
and so fot~th.
Unless defined otherwise, all tecluiical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing the
cell lines, protocols, reagents and vectors which are reported in the
publications and which might be
used in connection with various embodiments of the invention. Nothing herein
is to be construed as an
admission that the invention is not entitled to antedate such disclosure by
virtue of prior invention.
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DEFINITIONS
"INTSIG" refers to the amino acid sequences of substantially purified 1NTSIG
obtained from
any species, particularly a mammalian species, including bovine, ovine,
porcine, murine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
1NTSIG. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of 1NTSIG either by
directly interacting with
INTSIG or by acting on components of the biological pathway in which 1NTSIG
participates.
An "allelic variant" is an alternative form of the gene encoding INTSIG.
Allelic variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding INTSIG include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as INTSIG or a
polypeptide with at least one functional characteristic of 1NTSIG. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding INTSIG, and improper or unexpected hybridization
to allelic variants, with
a locus other than the normal chromosomal locus for the polynucleotide
encoding 1NTSIG. The
encoded protein may also be "altered," and may contain deletions, insertions,
or substitutions of amino
acid residues which produce a silent change and result in a functionally
equivalent INTSIG.
Deliberate amino acid substitutions may be made on the basis of one or more
similarities in polarity,
charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic
nature of the residues, as long
as the biological or immunological activity of INTSIG is retained. For
example, negatively charged
amino acids may include aspartic acid and glutamic acid, and positively
charged amino acids may
include lysine and arginine. Amino acids with uncharged polar side chains
having similar hydrophilicity
values may include: asparagine and glutamine; and serine and threonine. Amino
acids with uncharged
side chains having similar hydrophilicity values may include: leucine,
isoleucine, and valine; glycine and
alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" can refer to an oligopeptide,
a peptide, a
CA 02458645 2004-02-16
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polypeptide, or a protein sequence, or a fragment of any of these, and to
naturally occurring or
synthetic molecules. Where "amino acid sequence" is recited to refer to a
sequence of a naturally
occurring protein molecule, "amino acid sequence" and like terms are not meant
to limit the amino acid
sequence to the complete native amino acid sequence associated with the
recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid. Amplification
may be carried out using polymerase chain reaction (PCR) technologies or other
nucleic acid
amplification technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of INTSIG. Antagonists may include proteins such as antibodies, anticalins,
nucleic acids,
carbohydrates, small molecules, or any other compound or composition which
modulates the activity of
1NTSIG either by directly interacting with INTSIG or by acting on components
of the biological
pathway in which INTSIG participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind INTSIG polypeptides can be prepared using intact
polypeptides or using
fragments containing small peptides of interest as the immunizing antigen. The
polypeptide or
oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit)
can be derived from the
translation of RNA, or synthesized chemically, and can be conjugated to a
carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLI~. The coupled peptide is
then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen used
to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
specific molecular target. Aptamers are derived from an ira vitf°o
evolutionary process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large
combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may
include
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deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other
nucleotide-like molecules. The
nucleotide components of an aptamer may have modified sugar groups (e.g., the
2'-OH group of a
ribonucleotide may be replaced by 2 =F or 2'-NHz), which may improve a desired
property, e.g.,
resistance to nucleases or longer lifetime in blood. Aptamers may be
conjugated to other molecules,
e.g., a high molecular weight carrier to slow clearance of the aptamer from
the circulatory system.
Aptamers may be. specifically cross-linked to their cognate ligands, e.g., by
photo-activation of a
cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
The term "intramer" refers to an aptamer which is expressed ifa vivo. For
example, a
vaccinia virus-based RNA expression system has been used to express specific
RNA aptamers at
high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc.
Natl. Acad. Sci. USA
96:3606-3610).
The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or
other left-
handed nucleotide derivatives or nucleotide-like molecules. Aptamers
containing left-handed
nucleotides are resistant to degradation by naturally occurnng enzymes, which
normally act on
substrates containing right handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a polynucleotide having a specific nucleic acid sequence.
Antisense compositions
may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having
modified backbone .
linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates;
oligonucleotides
having modified sugar groups such as 2'-methoxyethyl sugars or 2'-
methoxyethoxy sugars; or
oligonucleotides having modified bases such as 5-methyl cytosine, 2'-
deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense molecules may be produced by any method including
chemical synthesis
or transcription. Once introduced into a cell, the complementary antisense
molecule base-pairs with a
naturally occurring nucleic acid sequence produced by the cell to form
duplexes which block either
transcription or translation. The designation "negative" or "minus" can refer
to the antisense strand,
and the designation "positive" or "plus" can refer to the sense strand of a
reference DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "imrnunogenic"
refers to the capability of the natural, recombinant, or synthetic )NTSIG, or
of any oligopeptide
thereof, to induce a specific immune response in appropriate animals or cells
and to bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
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sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5
A "composition comprising a given polynucleotide" and a "composition
comprising a given
polypeptide" can refer to any composition containing the given polynucleotide
or polypeptide. The
composition may comprise a dry formulation or an aqueous solution.
Compositions comprising
polynucleotides encoding INTSIG or fragments of 1NTSIG may be employed as
hybridization probes.
The probes may be stored in freeze-dried form and may be associated with a
stabilizing agent such as
a carbohydrate. In hybridizations, the probe may be deployed in an aqueous
solution containing salts
(e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other
components (e.g., Denhardt's
solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to
repeated DNA sequence analysis to resolve uncalled bases, extended using the
XL-PCR kit (Applied
Biosystems, Foster City CA) in the 5' and/or the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI) or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended
and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original a ino acid in a protein anal
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn
Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Asn, Glu, His
3o Glu
Asp, Gln, His
Gly Ala
~s Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
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Ser Cys, Thr
T~' Ser, Val
T~ Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains
at least one biological or immunological function of the natural molecule. A
derivative polypeptide is
one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(axons). Since an
axon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of INTSIG or a polynucleotide encoding INTSIG
which can
be identical in sequence to, but shorter in length than, the parent sequence.
A fragment may comprise
up to the entire length of the defined sequence, minus one nucleotide/amino
acid residue. For
example, a fragment may comprise from about 5 to about 1000 contiguous
nucleotides or amino acid
residues. A fragment used as a probe, primer, antigen, therapeutic molecule,
or for other purposes,
may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at
least 500 contiguous
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nucleotides or amino acid residues in length. Fragments may be preferentially
selected from certain
regions of a molecule. For example, a polypeptide fragment may comprise a
certain length of
contiguous amino acids selected from the brst 250 or 500 amino acids (or first
25% or 50%) of a
polypeptide as shown in a certain defined sequence. Clearly these lengths are
exemplary, and any
length that is supported by the specification, including the Sequence Listing,
tables, and figures, may be
encompassed by the present embodiments.
A fragment of SEQ ~ N0:46-90 can comprise a region of unique polynucleotide
sequence
that specifically identifies SEQ ll~ N0:46-90, for example, as distinct from
any other sequence in the
genome from which the fragment was obtained. A fragment of SEQ m N0:46-90 can
be employed
in one or more embodiments of methods of the invention, for example, in
hybridization and
amplification technologies and in analogous methods that distinguish SEQ m
NO:46-90 from related
polynucleotides. The precise length of a fragment of SEQ ll~ N0:46-90 and the
region of SEQ ID
N0:46-90 to which the fragment corresponds are routinely determinable by one
of ordinary skill in the
art based on the intended purpose for the fragment.
A fragment of SEQ ID N0:1-45 is encoded by a fragment of SEQ ll~ N0:46-90. A
fragment of SEQ ID N0:1-45 can comprise a region of unique amino acid sequence
that specifically
identifies SEQ ~ N0:1-45. For example, a fragment of SEQ ID N0:1-45 can be
used as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ ll~ N0:1-45.
The precise length of a fragment of SEQ ID NO:1-45 and the region of SEQ ID
N0:1-45 to which
the fragment corresponds can be determined based on the intended purpose for
the fragment using
one or more analytical methods described herein or otherwise known in the art.
A "full length" polynucleotide is one containing at least a translation
initiation codon (e.g., .
methionine) followed by an open reading frame and a translation termination
codon. A "full length"
polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer to
the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps in
the sequences being compared iu order to optimize alignment between two
sequences, and therefore
achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using one
or more
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computer algorithms or programs known in the art or described herein. For
example, percent identity
can be determined using the default parameters of the CLUSTAL V algorithm as
incorporated into
the MEGALIGN version 3.12e sequence alignment program. This program is part of
the
LASERGENE software package, a suite of molecular biological analysis programs
(DNASTAR,
Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989;
CABIOS 5:151-
153) and in Higgins, D.G. et al. (1992; CABIOS 8:189-191). For pairwise
alignments of
polynucleotide sequences, the default parameters are set as follows: Ktuple=2,
gap penalty=5,
window=4, and "diagonals saved"=4. The "weighted" residue weight table is
selected as the default.
Percent identity is reported by CLUSTAL V as the "percent similarity" between
aligned
polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
which can be used is provided by the National Center for Biotechnology
Information (NCBI) Basic
Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol.
Biol. 215:403-410), which
is available from several sources, including the NCBI, Bethesda, MD, and on
the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUN162
Reward for- match: 1
Penalty for mismatch: -2
Opeyi Gap: 5 arid Extension Gap: 2 peraalties
Gap x drop-off. 50
Expect: 10
3o Word Size: 1l
Filter-: o~Z
Percent identity may be measured over the length of an entire defined
sequence, for example,
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as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length supported
by the sequences shown herein, in the tables, figures, or Sequence Listing,
may be used to describe a
length over which percentage identity may be measured.
Nucleic acid sequences that do not show a lugh degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=S, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 aid Extension Gap: 1 peyaalties
Gap x drop-off 50
Expect: 10
Wof~d Size: 3
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Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length,
for example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody' refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability. .
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
2o stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill in
the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1% (w/v) SDS, and about 100 ~Cg/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al. (1989)
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Molecular Cloning: A Laborato~ Manual, 2nd ed., vol. 1-3, Cold Spring Harbor
Press, Plainview NY;
specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1% SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C may be used. SSC concentration may
be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
sheared and denatured salinon sperm DNA at about 100-200 ~Cg/ml. Organic
solvent, such as
formamide at a concentration of about 35-50%o v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acids by
virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex
may be formed in solution (e.g., Cot or Rot analysis) or formed between one
nucleic acid present in
solution and another nucleic acid immobilized on a solid support (e.g., paper,
membranes, filters, chips,
pins or glass slides, or any other appropriate substrate to which cells or
their nucleic acids have been
fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
polynucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of INTSIG
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment
of INTSIG which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, antibodies, or other chemical compounds on a substrate.
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The terms "element" and "array element" refer to a polynucleotide,
polypeptide, antibody, or
other chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of INTSIG. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
functional, or immunological properties of 7NTSIG.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terniinal lysine confers solubility
to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation, and
may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an INTSIG may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in the
art. These processes may occur synthetically or biochemically. Biochemical
modifications will vary
by cell type depending on the enzymatic milieu of INTSIG.
"Probe" refers to nucleic acids encoding INTSIG, their complements, or
fragments thereof,
which are used to detect identical, allelic or related nucleic acids. Probes
are isolated oligonucleotides
or polynucleotides attached to a detectable label or reporter molecule.
Typical labels include
radioactive isotopes, ligands, chemiluminescent agents, and enzymes. "Primers"
are short nucleic
acids, usually DNA oligonucleotides, which may be annealed to a target
polynucleotide by
complementary base-pairing. The primer may then be extended along the target
DNA strand by a
DNA polymerase enzyme. Primer pairs can be used for amplification (and
identification) of a nucleic
acid, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
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nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989; Molecular Cloning: A Laboratory Manual, 2"d
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY), Ausubel, F.M. et al. (1999; Short
Protocols in Molecular
Biology, 4''' ed., John Wiley & Sons, New York NY), and Innis, M. et al.
(1990; PCR Protocols, A
Guide to Methods and Applications, Academic Press, San Diego CA). PCR primer
pairs can be
derived from a known sequence, for example, by using computer programs
intended for that purpose
such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas
South West Medical Center, Dallas TX) is capable of choosing specific primers
from megabase
sequences and is thus useful for designing primers on a genome-wide scope. The
Primer3 primer
selection program (available to the public from the Whitehead Institute/MIT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
also be obtained from their respective sources and modified to meet the user's
specih.c needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
technologies, for example,
as PCR or sequencing primers, microarray elements, or specific probes to
identify fully or partially
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complementary polynucleotides in a sample of nucleic acids. Methods of
oligonucleotide selection are
not limited to those described above.
A "recombinant nucleic acid" is a nucleic acid that is not naturally occurring
or has a
sequence that is made by an artificial combination of two or more otherwise
separated segments of
sequence. This artificial combination is often accomplished by chenucal
synthesis or, more commonly,
by, the artificial manipulation of isolated segments of nucleic acids, e.g.,
by genetic engineering
techniques such as those described in Sambrook, supf-a. The term recombinant
includes nucleic acids
that have been altered solely by addition, substitution, or deletion of a
portion of the nucleic acid.
Frequently, a recombinant nucleic acid may include a nucleic acid sequence
operably linked to a
promoter sequence. Such a recombinant nucleic acid may be part of a vector
that is used, for
example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA molecule, is composed of the same
linear
sequence of nucleotides as the reference DNA molecule with the exception that
all occurrences of
the nitrogenous base thymine are replaced with uracil, and the sugar backbone
is composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing INTSIG,
nucleic acids encoding INTSIG, or fragments thereof may comprise a bodily
fluid; an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or cDNA,
in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
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synthetic binding composition. The interaction is dependent upon the presence
of a particular structure
of the protein, e.g., the antigenic determinant or epitope, recognized by the
binding molecule. For
example, if an antibody is specific for epitope "A," the presence of a
polypeptide comprising the
epitope A, or the presence of free unlabeled A, in a reaction containing free
labeled A and the
antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least about 60%
free, preferably at least about 75% free, and most preferably at least about
90% free from other
components with which they are naturally associated.
1o A "substitution" refers to the replacement of one or more amino acid
residues or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" or "expression profile" refers to the collective pattern
of gene expression
by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or viral
infection, electroporation, heat shock, lipofection, and particle bombardment.
The term "transformed
cells" includes stably transformed cells in which the inserted DNA is capable
of replication either as
an autonomously replicating plasmid or as part of the host chromosome, as well
as transiently
transformed cells which express the inserted DNA or RNA for limited periods of
time.
A "transgenic organism," as used herein, is any organism, including but not
limited to animals
and plants, in which one or more of the cells of the organism contains
heterologous nucleic acid
introduced by way of human intervention, such as by transgenic techniques well
known in the art. The
nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor of the cell,
by way of deliberate genetic manipulation, such as by microinjection or by
infection with a
recombinant virus. In another embodiment, the nucleic acid can be introduced
by infection with a
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recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002)
Science 295:868-872). The
term genetic manipulation does not include classical cross-breeding, or iyt
vitro fertilization, but rather
is directed to the introduction of a recombinant DNA molecule. The transgenic
organisms
contemplated in accordance with the present invention include bacteria,
cyanobacteria, fungi, plants
and animals. The isolated DNA of the present invention can be introduced into
the host by methods
known in the art, for example infection, transfection, transformation or
transconjugation. Techniques
for transferring the DNA of the present invention into such organisms are
widely known and provided
in references such as Sambrook et al. (1989), supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotides that vary from one
species to another. The
resulting polypeptides will generally have significant amino acid identity
relative to each other. A
polymorphic variant is a variation in the polynucleotide sequence of a
particular gene between
individuals of a given species. Polymorphic variants also may encompass
"single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
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sequence identity over a certain defined length of one of the polypeptides..
THE INVENTION
Various embodiments of the invention include new human intracellular signaling
molecules
(1NTSIG), the polynucleotides encoding ll~TSIG, and the use of these
compositions for the diagnosis,
treatment, or prevention of cell proliferative, endocrine,
autoimmune/inflammatory, neurological,
gastrointestinal, reproductive, developmental, and vesicle trafficking
disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
embodiments of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to
a single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is
denoted by both a polypeptide sequence identification number (Polypeptide SEQ
~ NO:) and an
Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown. Each
polynucleotide .
sequence is denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ ID
NO:) and an Incyte polynucleotide consensus sequence number (Incyte
Polynucleotide H~) as shown.
Column 6 shows the Incyte ID numbers of physical, full length clones
corresponding to the polypeptide
and polynucleotide sequences of the invention. The full length clones encode
polypeptides which have
at least 9~~/o sequence identity to the polypeptide sequences shown in column
3.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database and the PROTEOME
database.
Columns 1 and 2 show the polypeptide sequence identification number
(Polypeptide SEQ ID NO:) and
the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID)
for polypeptides of the
invention. Column 3 shows the GenBank identification number (GenBank )D NO:)
of the nearest
GenBank homolog and the PROTEOME database identification numbers (PROTEOME H~
NO:) of
the nearest PROTEOME database homologs. Column 4 shows the probability scores
for the matches
between each polypeptide and its homolog(s). Column 5 shows the annotation of
the GenBank and
PROTEOME database homolog(s) along with relevant citations where applicable,
all of which are
expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1 and
2 show the polypeptide sequence identification number (SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of
the invention. Column
3 shows the number of amino acid residues in each polypeptide. Column 4 shows
potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the MOTIFS
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program of the GCG sequence analysis software package (Genetics Computer
Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains,
and motifs. Column 7
shows analytical methods for protein structure/function analysis and in some
cases, searchable
databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are GTPase-associated
proteins. For example, SEQ
ID N0:1 is 53 % identical, from residue 8190 to residue E706, to human guanine
nucleotide regulatory
protein (GenBank ID g484102) as determined by the Basic Local Alignment Search
Tool (BLAST).
(See Table 2.) The BLAST probability score is 1.3e-129, which indicates the
probability of obtaining
the observed polypeptide sequence alignment by chance. SEQ ID NO:1 also
contains a PH domain, a
RhoGEF domain, and an SH3 domain as determined by searching for statistically
significant matches
in the hidden Markov model (H1VIIVI)-based PFAM database of conserved protein
family domains.
(See Table 3.) Data from additional BLAST analyses provide further
corroborative evidence that
SEQ m NO:1 is a guanine nucleotide regulatory protein.
As another example, SEQ lD N0:6 is 58% identical, from residue L225 to residue
C1845, to
human nuclear dual-specificity phosphatase (GenBank ll~ g3015538) as
determined by BLAST. The
BLAST probability score is 0Ø SEQ 1D N0:2 also contains DENN (AEX-3) and PH
domains as
determined by searching for statistically significant matches in the hidden
Markov model (HMM)-
based PFAM database. Data from further BLAST analyses provide corroborative
evidence that SEQ
ID NO:6 is a dual-specificity phosphatase.
As another example, SEQ ll7 NO:10 is 99% identical, from residue A44 to
residue M316, to
human TRAF4 associated factor 1 (GenBank 1D g4580011) as determined by BLAST.
The BLAST
probability score is 1.0e-138. In addition, SEQ ID NO:10 is 50% identical,
from residue M18 to
residue V775, to murine semaphorin cytoplasmic domain-associated protein 3B
(GenBank ID
g6651021) as determined by BLAST. The BLAST probability score is 9.6e-51. SEQ
ID N0:10 also
contains a PDZ domain as determined by searching for statistically significant
matches in the hidden
Markov model (HMM)-based PFAM database. Data from BLAST-PRODOM, BLllVIPS, and
MOT1FS analyses provide further corroborative evidence that SEQ ID NO:10 is a
signal transduction
molecule.
3o As another example, SEQ lD NO:15 is 79% identical, from residue M1 to
residue L917, to
mouse PDZ-RGS3 protein, (GenBank ID g13774477) as determined by BLAST. The
BLAST
probability score is 0Ø The PDZ-RGS3 protein, binds B ephrins through a PDZ
domain, and has a
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regulator of heterotrimeric G protein signaling (RGS) domain (Lu,Q. et al.
(2001) Cell 105 (1), 69-79).
SEQ ID N0:15 also contains a regulator of G protein signaling domain and a PDZ
domain as
determined by searching for statistically significant matches in the hidden
Markov model (HMM)-
based PFAM database. Data from BLM'S, MOTIFS, and further BLAST analyses
provide
corroborative evidence that SEQ ID N0:15 is a PDZ-RGS3 protein.
As another example, SEQ ID N0:24 is 91% identical, from residue M1 to residue
D1023, to
p116Rip (GenBank ID g1657837), a Rho-interacting GDP/GTP exchange factor, as
determined by
BLAST. The BLAST probability score is 0Ø SEQ ID N0:24 also contains a PH
domain as
determined by searching for statistically significant matches in the hidden
Markov model (I~~VI)-
based PFAM database. Data from additional BLAST analysis provide further
corroborative evidence
that SEQ ID NO:24 is a Rho-binding protein.
As another example, SEQ ID NO:27 is 82% identical, from residue P56 to residue
L1123, to
the sorbin and SH3 domain-containing gene (GenBank ID g13650131) as determined
by BLAST. The
BLAST probability score is 0.0, which indicates the probability of obtaining
the observed polypeptide
sequence alignment by chance. SEQ ID N0:27 contains an SH3 and a sorbin domain
as determined
by searching for statistically significant matches in the hidden Markov model
(HMM)-based PFAM
database. Data from BLM'S and BLAST analyses provide further corroborative
evidence that SEQ
ll7 N0:27 is an SH3 domain-containing protein.
As another example, SEQ ll~ NO:30, SEQ ID N0:32-36, and SEQ ID N0:39 have
significant
homology to Rattus norve 'cus synaptic ras GTPase-activating protein SynGAP
(GenBank ~
g2935448), as determined by BLAST. SEQ ID N0:30 is 95% identical to GenBank ID
g2935448
from residue M1 to residue P1143. SEQ ID N0:32 is 97% identical to GenBank ll~
82935448 from
residue M1 to residue V1308. SEQ ID N0:33 is 99% identical to GenBank ID
82935448 from
residue M1 to residue V1279. SEQ ID N0:34 is 99% identical to GenBank ID
82935448 from
residue M1 to residue V1293. SEQ lD N0:35 is 99% identical to GenBank ID
82935448 from
residue M1 to residue L387 and 98% identical from residue V416 to residue
P1157. SEQ ll~ N0:36
is 98% identical to GenBank ID 82935448 from residue M1 to residue P1128. SEQ
ID N0:39 is 99%
identical to GenBank ll~ 82935448 from residue M1 to residue L545 and 98%
identical from residue
V574 to residue V1322. (See Table 2.) The BLAST probability score for each of
SEQ ll~ N0:30,
SEQ ID N0:32-36, and SEQ ll~ N0:39 is 0.0, which indicates the probability of
obtaining the
observed polypeptide sequence alignments by chance. SEQ ID N0:30, SEQ 1D N0:32-
36, and SEQ
ID N0:39 are identified as GTPase activating proteins, as determined by BLAST
analysis using the
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PROTEOME database. SEQ ID NO:30, SEQ ID N0:32-36, and SEQ ID N0:39 each
contain a Ras
GTPase-activating proteins signature and profile domain as determined by
searching for statistically
significant matches in the hidden Markov model (F~~IM)-based PFAM database.
(See Table 3.)
Data from BLllVIPS, MOTIFS, and PROF1L,ESCAN analyses provide further
corroborative evidence
that SEQ ID N0:30, SEQ ll~ N0:32-36, and SEQ ll~ N0:39 are GTPase activating
proteins.
As another example, SEQ ID N0:42 is 97% identical, from residue M33 to residue
S309, to
human Ras like GTPase (GenBank ll~ g2117166) as determined by BLAST. The BLAST
probability
score is 4.5e-145. SEQ ID NO:42 is a GTP-binding protein, as determined by
BLAST analysis using
the PROTEOME database. SEQ ID N0:42 also contains a Ras family domain as
determined by
searching for statistically significant matches in the hidden Markov model
(IEVVIM)-based PFAM
database. Data from BLllVIPS, MOTIFS and additional BLAST analyses provide
further
corroborative evidence that SEQ ID N0:42 is a Ras family GTPase. SEQ ID N0:2-
5, SEQ ID
N0:7-9, SEQ ID NO:11-14, SEQ ID N0:16-23, SEQ ll~ NO:25-26, SEQ ID N0:28-29,
SEQ ID
N0:31, SEQ ID N0:37-38, and SEQ ll~ NO:40-41 were analyzed and annotated in a
similar manner.
The algorithms and parameters for the analysis of SEQ 117 N0:1-45 are
described in Table 7.
As shown in Table 4, the full length polynucleotide embodiments were assembled
using cDNA
sequences or coding (exon) sequences derived from genomic DNA, or any
combination of these two
types of sequences. Column 1 lists the polynucleotide sequence identification
number (Polynucleotide
SEQ ll~ NO:), the corresponding Incyte polynucleotide consensus sequence
number (Incyte ID) for
each polynucleotide of the invention, and the length of each polynucleotide
sequence in basepairs.
Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA
and/or genomic
sequences used to assemble the full length polynucleotide embodiments, and of
fragments of the
polynucleotides which are useful, for example, in hybridization or
amplification technologies that
identify SEQ ID N0:46-90 or that distinguish between SEQ ID N0:46-90 and
related polynucleotides.
The polynucleotide fragments described in Column 2 of Table 4 may refer
specifically, for
example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from
pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2
may refer to GenBank
cDNAs or ESTs which contributed to the assembly of the full length
polynucleotides. In addition, the
polynucleotide fragments described in column 2 may identify sequences derived
from the ENSEMBL
(The Sanger Centre, Cambridge, UK) database (i.e., those sequences including
the designation
"ENST"). Alternatively, the polynucleotide fragments described in column 2 may
be derived from the
NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences
including the
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designation "NM" or "NT") or the NCBI RefSeq Protein Sequence Records (i. e.,
those sequences
including the designation "NP"). Alternatively, the polynucleotide fragments
described in column 2
may refer to assemblages of both cDNA and Genscan-predicted axons brought
together by an "axon
stitching" algorithm. For example, a polynucleotide sequence identified as
FL XXXXXX NI IVZ YYYYI' N3 N4 represents a "stitched" sequence in which ~:XX
is the
identification number of the cluster of sequences to which the algorithm was
applied, and is the
number of the prediction generated by the algorithm, and N1,2,3...~ if
present, represent specific axons
that may have been manually edited during analysis (See Example V).
Alternatively, the
polynucleotide fragments in column 2 may refer to assemblages of axons brought
together by an
"axon-stretching" algorithm. For example, a polynucleotide sequence identified
as
FLXX~XXX ~AAAAA_gBBBBB_1 1V is a "stretched" sequence, withXXX~'XX being the
Incyte
project identification number, gAAAAA being the GenBank identification number
of the human
genomic sequence to which the "axon-stretching" algorithm was applied, gBBBBB
being the GenBank
identification number or NCBI RefSeq identification number of the nearest
GenBank protein homolog,
and N referring to specific axons (See Example V). In instances where a RefSeq
sequence was used
as a protein homolog for the "axon-stretching" algorithm, a RefSeq identifier
(denoted by "NM,"
"NP," or "NT") may be used in place of the GenBank identifier (i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs
GNN, GFG,Exon prediction from genomic sequences using,
for example,
ENST GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK)
GBI Hand-edited analysis of genomic sequences.
Stitched or stretched genomic sequences (see
Example V).
INCY Full length transcript and axon prediction
from mapping of EST
sequences to the genome. Genomic location
and EST composition
data are combined to predict the axons and
resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in
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Table 4 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant Incyte
cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotides which
were assembled using Incyte cDNA sequences. The representative cDNA library is
the Incyte
cDNA library which is most frequently represented by the Incyte cDNA sequences
which were used
to assemble and confirm the above polynucleotides. The tissues and vectors
which were used to
construct the cDNA libraries shown in Table 5 are described in Table 6.
Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide
sequences of
the invention, along with allele frequencies in different human populations.
Columns 1 and 2 show the
polynucleotide sequence identification number (SEQ ID NO:) and the
corresponding Incyte project
identification number (PID) for polynucleotides of the invention. Column 3
shows the Incyte
identification number for the EST in which the SNP was detected (EST ID), and
column 4 shows the
identification number for the SNP (SNP D7). Column 5 shows the position within
the EST sequence
at which the SNP is located (EST SNP), and column 6 shows the position of the
SNP within the full-
length polynucleotide sequence (CB1 SNP). Column 7 shows the allele found in
the EST sequence.
Columns 8 and 9 show the two alleles found at the SNP site. Column 10 shows
the amino acid
encoded by the codon including the SNP site, based upon the allele found in
the EST. Columns 11-14
show the frequency of allele 1 in four different human populations. An entry
of n/d (not detected)
indicates that the frequency of allele 1 in the population was too low to be
detected, while nla (not
available) indicates that the allele frequency was not determined for the
population.
The invention also encompasses INTSIG variants. A preferred INTSIG variant is
one which
has at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the INTSIG amino acid sequence, and which contains at
least one functional or
structural characteristic of 1NTSIG.
Various embodiments also encompass polynucleotides which encode INTSIG. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:46-90, which encodes INTSIG. The
polynucleotide
sequences of SEQ ID N0:46-90, as presented in the Sequence Listing, embrace
the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced
with uracil, and the
sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses variants of a polynucleotide encoding INTSIG.
In particular,
such a variant polynucleotide will have at least about 70%, or alternatively
at least about 85%, or even
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at least about 95% polynucleotide sequence identity to a polynucleotide
encoding lNTSIG. A
particular aspect of the invention encompasses a variant of a polynucleotide
comprising a sequence
selected from the group consisting of SEQ )D N0:46-90 which has at least about
70%, or alternatively
at least about 85%, or even at least about 95% polynucleotide sequence
identity to a nucleic acid
sequence selected from the group consisting of SEQ D7 N0:46-90. Any one of the
polynucleotide
variants described above can encode a polypeptide which contains at least one
functional or structural
characteristic of INTSIG.
In addition, or in the alternative, a polynucleotide variant of the invention
is a splice variant of a
polynucleotide encoding )N'TSIG. A splice variant may have portions which have
significant sequence
identity to a polynucleotide encoding INTSIG, but will generally have a
greater or lesser number of
polynucleotides due to additions or deletions of blocks of sequence arising
from alternate splicing of
exons during mRNA processing. A splice variant may.have less than about 70%,
or alternatively less
than about 60%, or alternatively less than about 50% polynucleotide sequence
identity to a
polynucleotide encoding 1NTSIG over its entire length; however, portions of
the splice variant will
have at least about 70%, or alternatively at least about 85%, or alternatively
at least about 95%, or
alternatively 100% polynucleotide sequence identity to portions of the
polynucleotide encoding
INTSIG. For example, a polynucleotide comprising a sequence of SEQ ~ N0:54 and
a
polynucleotide comprising a sequence of SEQ ID NO:90 are splice variants of
each other; a
polynucleotide comprising a sequence of SEQ ID N0:57 and a polynucleotide
comprising a sequence
of SEQ ll~ N0:59 are splice variants of each other; a polynucleotide
comprising a sequence of SEQ
lD N0:69 and a polynucleotide comprising a sequence of SEQ )D N0:70 are splice
variants of each
other; a polynucleotide comprising a sequence of SEQ ID N0:75, a
polynucleotide comprising a
sequence of SEQ ll~ N0:77, a polynucleotide comprising a sequence of SEQ ID
N0:78, a
polynucleotide comprising a sequence of SEQ )D NO:79, a polynucleotide
comprising a sequence of
SEQ lD NO:80, a polynucleotide comprising a sequence of SEQ ID N0:81, and a
polynucleotide
comprising a sequence of SEQ ll~ N0:84 are splice variants of each other; and
a polynucleotide
comprising a sequence of SEQ ~ N0:76 and a polynucleotide comprising a
sequence of SEQ ID
N0:88 are splice variants of each other. Any one of the splice variants
described above can encode a
polypeptide which contains at least one functional or structural
characteristic of INq'SIG.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
genetic code, a multitude of polynucleotide sequences encoding INTSIG, some
bearing minim__al
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
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produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring INTSIG, and all such variations
are to be considered as
being specifically disclosed.
Although polynucleotides which encode 1NTSIG and its variants are generally
capable of
hybridizing to polynucleotides encoding naturally occurring INTSIG under
appropriately selected
conditions of stringency, it may be advantageous to produce polynucleotides
encoding INTSIG or its
derivatives possessing a substantially different codon usage, e.g., inclusion
of non-naturally occurring
codons. Codons may be selected to increase the rate at which expression of the
peptide occurs in a
particular prokaryotic or eukaryotic host in accordance with the frequency
with which particular
codons are utilized by the host. Other reasons for substantially altering the
nucleotide sequence
encoding INTSIG and its derivatives without altering the encoded amino acid
sequences include the
production of RNA transcripts having more desirable properties, such as a
greater half life, than
transcripts produced from the naturally occurring sequence.
The invention also encompasses production of polynucleotides which encode
INTSIG and
INTSIG derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the
synthetic polynucleotide may be inserted into any of the many available
expression vectors and cell
systems using reagents well known in the art. Moreover, synthetic chemistry
may be used to
introduce mutations into a polynucleotide encoding INTSIG or any fragment
thereof.
Embodiments of the invention can also include polynucleotides that are capable
of hybridizing
to the claimed polynucleotides, and, in particular, to those having the
sequences shown in SEQ ID
NO:46-90 anal fragments thereof, under various conditions of stringency (Wahl,
G.M. and S.L. Berger
(1987) Methods Enzymol. 152:399-407; IKimmel, A.R. (1987) Methods Enzymol.
152:507-511).
Hybridization conditions, including annealing and wash conditions, are
described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQLTENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ),
or combinations
of polymerases and proofreading exonucleases such as those found in the
ELONGASE amplification
system (Invitrogen, Carlsbad CA). Preferably, sequence preparation is
automated with machines
such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200
thermal cycler
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(MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied
Biosystems).
Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing
system (Applied
Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences),
or other
systems known in the art. The resulting sequences are analyzed using a variety
of algorithms which
are well known in the art (Ausubel et al., supf~a, ch. 7; Meyers, R.A. (1995)
Molecular Biolo~~
Biotechnology, Wiley VCH, New York NY, pp. 856-853).
The nucleic acids encoding INTSIG may be extended utilizing a partial
nucleotide sequence
and employing various PCR based methods known in the art to detect upstream
sequences, such as
promoters and regulatory elements. For example, one method which may be
employed, restriction-site
PCR, uses universal and nested primers to amplify unknown sequence from
genomic DNA within a
cloning vector (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). Another
method, inverse PCR,
uses primers that extend in divergent directions to amplify unknown sequence
from a circularized
template. The template is derived from restriction fragments comprising a
known genomic locus and
surrounding sequences (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186).
A third method, capture
PCR, involves PCR amplification of DNA fragments adjacent to known sequences
in human and
yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods
Applic. 1:111-119). In
this method, multiple restriction enzyme digestions and ligations may be used
to insert an engineered
double-stranded sequence into a region of unknown sequence before performing
PCR. Other
a
methods which may be used to retrieve unknown sequences are known in the art
(Parker, J.D. et al.
(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested
primers, and
PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genornic DNA. This
procedure
avoids the need to screen libraries and is useful in finding intron/exon
junctions. For all PCR-based
methods, primers may be designed using commercially available software, such
as OLIGO 4.06
primer analysis software (National Biosciences, Plymouth MN) or another
appropriate program, to be
about 22 to 30 nucleotides in length, to have a GC content of about 50% or
more, and to anneal to the
template at temperatures of about 68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
53
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the size or conErm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotides or fragments thereof
which encode
7NTSIG may be cloned in recombinant DNA molecules that direct expression of
INTSIG, or
fragments or functional equivalents thereof, in appropriate host cells. Due to
the inherent degeneracy
of the genetic code, other polynucleotides which encode substantially the same
or a functionally
equivalent polypeptides may be produced and used to express INTSIG.
The polynucleotides of the invention can be engineered using methods generally
known in the
art in order to alter INTSIG-encoding sequences for a variety of purposes
including, but not limited to,
modification of the cloning, processing, and/or expression of the gene
product. DNA shuffling by
random fragmentation and PCR reassembly of gene fragments and synthetic
oligonucleotides may be
used to engineer the nucleotide sequences. For example, oligonucleotide-
mediated site-directed
mutagenesis may be used to introduce mutations that create new restriction
sites, alter glycosylation
patterns, change codon preference, produce splice variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or improve
the biological properties of 1NTSIG, such as its biological or enzymatic
activity or its ability to bind to
other molecules or compounds. DNA shuffling is a process by which a library of
gene variants is
produced using PCR-mediated recombination of gene fragments. The library is
then subjected to
selection or screening procedures that identify those gene variants with the
desired properties. These
preferred variants may then be pooled and further subjected to recursive
rounds of DNA shuffling and
selection/screeniug. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular
evolution. For example, fragments of a single gene containing random point
mutations may be
recombined, screened, and then reshuffled until the desired properties are
optimized. Alternatively,
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fragments of a given gene may be recombined with fragments of homologous genes
in the same gene
family, either from the same or different species, thereby maximizing the
genetic diversity of multiple
naturally occurring genes in a directed and controllable manner.
In another embodiment, polynucleotides encoding INTSIG may be synthesized, in
whole or in
part, using one or more chemical methods well known in the art (Caruthers,
M.H. et al. (1980)
Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp.
Ser. 7:225-232).
Alternatively, INTSIG itself or a fragment thereof may be synthesized using
chemical methods known
in the art. For example, peptide synthesis can be performed using various
solution-phase or
solid-phase techniques (Creighton, T. (1984) Proteins, Structures and
Molecular Properties,- WII
Freeman, New York NY, pp. 55-60; Roberge, J.Y. et al. (1995) Science 269:202-
204). Automated
synthesis may be achieved using the ABI 431A peptide synthesizer (Applied
Biosystems).
Additionally, the amino acid sequence of 1N'I'SIG, or any part thereof, may be
altered during direct
synthesis and/or combined with sequences from other proteins, or any part
thereof, to produce a
variant polypeptide or a polypeptide having a sequence of a naturally
occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography (Chiez, R.M. and F.2. Regnier (1990) Methods Enzymol. 182:392-
421). The
composition of the synthetic peptides may be confirmed by amino acid analysis
or by sequencing
(Creighton, supra, pp. 28-53).
In order to express a biologically active 1NTSIG, the polynucleotides encoding
INTSIG or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which contains
the necessary elements for trauscriptional and translational control of the
inserted coding sequence in
a suitable host. These elements include regulatory sequences, such as
enhancers, constitutive and
inducible promoters, and 5' and 3'untranslated regions in the vector and in
polynucleotides encoding
1NTSIG. Such elements may vary in their strength and specificity. Specific
initiation signals may also
be used to achieve more efficient translation of polynucleotides encoding
INTSIG. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where a
polynucleotide sequence encoding INTSIG and its initiation codon and upstream
regulatory sequences
are inserted into the appropriate expression vector, no additional
transcriptional or translational control
signals may be needed. However, in cases where only coding sequence, or a
fragment thereof, is
inserted, exogenous translational control signals including an in-frame ATG
initiation codon should be
provided by the vector. Exogenous translational elements and initiation codons
may be of various
origins, both natural and synthetic. The efficiency of expression may be
enhanced by the inclusion of
CA 02458645 2004-02-16
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enhancers appropriate for the particular host cell system used (Scharf, D. et
al. (1994) Results Probl.
Cell Differ. 20:125-162).
Methods,which are well known to those skilled in the art may be used to
construct expression
vectors containing polynucleotides encoding INTSIG and appropriate
transcriptional and translational
control elements. These methods include in vitf-o recombinant DNA techniques,
synthetic techniques,
and ih vivo genetic recombination (Sambrook, J. et al. (1989) Molecular
Cloning A LaboratorX
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel
et al., supra, ch. 1, 3,
and 15).
A variety of expression vector/host systems may be utilized to contain and
express
polynucleotides encoding INTSIG. These include, but are not limited to,
microorganisms such as
bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA
expression vectors;
yeast transformed with yeast expression vectors; insect cell systems infected
with viral expression
vectors (e.g., baculovirus); plant cell systems transformed with viral
expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with
bacterial expression vectors
(e.g., Ti or pBR322 plasmids); or animal cell systems (Sambrook, supra;
Ausubel et al., supra; Van
Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard,
E.K. et al. (1994)
Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene
Ther. 7:1937-1945;
Takamatsu, N. (1987) EMBO J. 6:307-311; The McGraw Hill Yearbook of Science
and Technolo~y
(1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984)
Proc. Natl. Acad.
Sci. USA 81:3655-3659; Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355).
Expression vectors
derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or
from various bacterial
plasmids, may be used for delivery of polynucleotides to the targeted organ,
tissue, or cell population
(Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu, M. et al. (1993)
Proc. Natl. Acad. Sci.
USA 90:6340-6344; Buller, R.M. et al. (1985) Nature 317:813-815; McGregor,
D.P. et al. (1994) Mol.
Tm_m__unol. 31:219-226; Verma, LM. and N. Somia (1997) Nature 389:239-242).
The invention is not
limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotides encoding INTSIG. For example,
routine cloning, subcloning,
and propagation of polynucleotides encoding 1NTSIG can be achieved using a
multifunctional E. coli
vector such as PBLUESCR1PT (Stxatagene, La Jolla CA) or PSPORT1 plasmid
(Invitrogen).
Ligation of polynucleotides encoding INTSIG into the vector's multiple cloning
site disrupts the lacZ
gene, allowing a colorimetric screening procedure for identification of
transformed bacteria containing
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recombinant molecules. In addition, these vectors may be useful for in vitro
transcription, dideoxy
sequencing, single strand rescue with helper phage, and creation of nested
deletions in the cloned
sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-
5509). When large
quantities of INTSIG are needed, e.g. for the production of antibodies,
vectors which direct high level
_ expression of 11VTSIG may be used. For example, vectors containing the
strong, inducible SP6 or T7
bacteriophage promoter may be used.
Yeast expression systems may be used for production of 7NTSIG. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Sacchar~ornyces ce~evisiae or Pichia
pastof-is. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign polynucleotide sequences into the host genome for stable
propagation (Ausubel et al.,
supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, C.A.
et al. (1994)
Bio/Technology 12:181-184).
Plant systems may also be used for expression of INTSIG. Transcription of
polynucleotides
encoding INTSIG may be driven by viral promoters, e.g., the 355 and 19S
promoters of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Brogue,
R. et al. (1984)
Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ.
17:85-105). These constructs
can be introduced into plant cells by direct DNA transformation or pathogen-
mediated transfection
(The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New
York NY, pp.
191-196).
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, polynucleotides encoding
1NTSIG may be
ligated into an adenovirus transcription/translation complex consisting of the
late promoter and tripartite
leader sequence. Insertion in a non-essential E1 or E3 region of the viral
genome may be used to
obtain infective virus which expresses INTSIG in host cells (Logan, J. and T.
Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659). In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
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constructed and delivered via conventional delivery methods (liposomes,
polycationic amino polymers,
or vesicles) for therapeutic purposes (Harnngton, J.J. et al. (1997) Nat.
Genet. 15:345-355).
For long term production of recombinant proteins in mammalian systems, stable
expression of
INTSIG in cell lines is preferred. For example, polynucleotides encoding
INTSIG can be transformed
into cell lines using expression vectors which may contain viral origins of
replication and/or
endogenous expression elements and a selectable marker gene on the same or on
a separate vector.
Following the introduction of the vector, cells may be allowed to grow for
about 1 to 2 days in enriched
media before being switched to selective media. The purpose of the selectable
marker is to confer
resistance to a selective agent, and its presence allows growth and recovery
of cells which
successfully express the introduced sequences. Resistant clones of stably
transformed cells may be
pf opagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk and apr-~ cells, respectively
(Wigler, M. et al. (1977)
Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823). Also,
antimetabolite, antibiotic, or herbicide
resistance can be used as the basis for selection. For example, eih, f~
confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively (Wigler, M. et al.
(1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al.
(1981) J. Mol. Biol.
150:1-14). Additional selectable genes have been described, e.g., trpB and
hisD, which alter cellular
requirements for metabolites (Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA
85:8047-8051). Visible markers, e.g., authocyanins, green fluorescent proteins
(GFP; Clontech), (3-
glucuronidase and its substrate (3-glucuronide, or luciferase and its
substrate luciferin may be used.
These markers can be used not only to identify transformants, but also to
quantify the amount of
transient or stable protein expression attributable to a specific vector
system (Rhodes, C.A. (1995)
Methods Mol. Biol. 55:121-131).
Although the presence/absence of marker gene expression suggests that the gene
of interest
is also present, the presence and expression of the gene may need to be
confirmed. For example, if
the sequence encoding INTSIG is inserted within a marker gene sequence,
transformed cells
containing polynucleotides encoding 1NTSIG can be identified by the absence of
marker gene
function. Alternatively, a marker gene can be placed in tandem with a sequence
encoding INT'SIG
under the control of a single promoter. Expression of the marker gene in
response to induction or
5s
CA 02458645 2004-02-16
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selection usually indicates expression of the tandem gene as well.
In general, host cells that contain the polynucleotide encoding 1NTSIG and
that express
1NTSIG may be identified by a variety of procedures known to those of skill in
the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations,
PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane, solution, or
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
T_mmunologi.cal methods for detecting and measuring the expression of- -
INT'SIG using either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FAGS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on INTSIG is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art
(Hampton, R. et al. (1990) Serological Methods a Laboratory Manual,- APS
Press, St. Paul MN, Sect..
IV; Coligan, J.E. et al. (1997) Current Protocols in Tm_m__unolo y, Greene
Pub. Associates and Wiley-
Interscience, New York NY; Pound, J.D. (1998) Tm_m_unochemical Protocols,
Humana Press, Totowa
NJ).
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled hybridization
or PCR probes for detecting sequences related to polynucleotides encoding
INTSIG include
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, polynucleotides encoding INTSIG, or any fragments thereof, may
be cloned into a
vector for the production of an mRNA probe. Such vectors are known in the art,
are commercially
available, and may be used to synthesize RNA probes in_vitr-o by addition of
an appropriate RNA
polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures
may be conducted
using a variety of commercially available kits, such as those provided by
Amersham Biosciences,
Promega (Madison WI), and US Biochemical. Suitable reporter molecules or
labels which may be
used for ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or
chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic
particles, and the like.
Host cells transformed with polynucleotides encoding INTSIG may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors containing
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polynucleotides which encode INTSIG may be designed to contain signal
sequences which direct
secretion of 1NTSIG through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted polynucleotides or to process the expressed protein in the desired
fashion. Such modifications
of the polypeptide include, but are not limited to, acetylation,
carboxylation, glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCI~, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
polynucleotides
encoding 1NTSIG may be ligated to a heterologous sequence resulting in
translation of a fusion protein
in any of the aforementioned host systems. For example, a chimeric INTSIG
protein containing a
heterologous moiety that can be recognized by a commercially available
antibody may facilitate the
screening of peptide libraries for inhibitors of INTSIG activity. Heterologous
protein and peptide
moieties may also facilitate purification of fusion proteins using
commercially available affinity
matrices. Such moieties include, but are not limited to, glutathione S-
transferase (GST), maltose
binding protein (MBP), thioredoxin (Trx), cahnodulin binding peptide (CBP), 6-
His, FLAG, c-myc, and
hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their
cognate fusion
proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
and metal-chelate resins,
respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity
purification of fusion
proteins using commercially available monoclonal and polyclonal antibodies
that specifically recognize
these epitope tags. A fusion protein may also be engineered to contain a
proteolytic cleavage site
located between the 1NTSIG encoding sequence and the heterologous protein
sequence, so that
1NTSIG may be cleaved away from the heterologous moiety following
purification. Methods for
fusion protein expression and purification are discussed in Ausubel et al.
(supf~a, ch. 10 and 16). A
variety of commercially available kits may also be used to facilitate
expression and purification of
fusion proteins.
In another embodiment, synthesis of radiolabeled 1NTSIG may be achieved ifa
vitt~o using the
TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These
systems couple
transcription and translation of protein-coding sequences operably associated
with the T7, T3, or SP6
CA 02458645 2004-02-16
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promoters. Translation takes place in the presence of a radiolabeled amino
acid precursor, for
example, 35S-methioni_ne.
INTSIG, fragments of INTSIG, or variants of INTSIG may be used to screen for
compounds
that specifically bind to INTSIG. One or more test compounds may be screened
for specific binding
to INTSIG. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test
compounds can be
screened for specific binding to INTSIG. Examples of test compounds can
include antibodies,
anticalins, oligonucleotides, proteins (e.g., ligands or receptors), or small
molecules.
In related embodiments, variants of INTSIG can be used to screen for binding
of test
compounds, such as antibodies, to INTSIG, a variant of INTSIG, or a
combination of 1NTSIG and/or
one or more variants INTSIG. In an embodiment, a variant of INTSIG can be used
to screen for
compounds that bind to a variant of INTSIG, but not to 1NTSIG having the exact
sequence of a
sequence of SEQ ID N0:1-45. 1NTSIG variants used to perform such screening can
have a range of
about 50% to about 99% sequence identity to INTSIG, with various embodiments
having 60%, 70%,
75%, 80%, 85%, 90%, and 95% sequence identity.
In an embodiment, a compound identified in a screen for specific binding to
1NTSIG can be
closely related to the natural ligand of INTSIG, e.g., a ligand or fragment
thereof, a natural substrate, a
structural or functional mimetic, or a natural binding partner (Coligan, J.E.
et al. (1991) Current
Protocols in Itnmunolo~y 1(2):Chapter 5). In another embodiment, the compound
thus identified can
be a natural ligand of a receptor INTSIG (Howard, A.D. et al. (2001) Trends
Pharmacol. Sci.22:132-
140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
In other embodiments, a compound identified in a screen for specific binding
to 1N'I'SIG can
be closely related to the natural receptor to which 1NTSIG binds, at least a
fragment of the receptor,
or a fragment of the receptor including all or a portion of the ligand binding
site or binding pocket. For
example, the compound may be a receptor for INTSIG which is capable of
propagating a signal, or a
decoy receptor for INTSIG which is not capable of propagating a signal
(Ashkenazi, A. and V.M.
Divit (1999) Curr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al. (2001)
Trends Tm_m__unol. 22:328-
336). The compound can be rationally designed using known techniques. Examples
of such
techniques include those used to construct the compound etanercept (ENBREL;
Amgen Inc.,
Thousand Oaks CA), which is efficacious for treating rheumatoid arthritis in
humans. Etanercept is
an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc
portion of human IgGl
(Taylor, P.C. et al. (2001) Curr. Opin. Tmm__unol. 13:611-616).
In one embodiment, two or more antibodies having similar or, alternatively,
different
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specificities can be screened for specific binding to INTSIG, fragments of
INTSIG, or variants of
INTSIG. The binding specificity of the antibodies thus screened can thereby be
selected to identify
particular fragments or variants of 7NT5IG. In one embodiment, an antibody can
be selected such
that its binding specificity allows for preferential identification of
specific fragments or variants of
1NTSIG. In another embodiment, an antibody can be selected such that its
binding specificity allows
for preferential diagnosis of a specific disease or condition having
increased, decreased, or otherwise
abnormal production of INTSIG.
In an embodiment, anticalins can be screened for specific binding to INTSIG,
fragments of
INTSIG, or variants of INTSIG. Anticalins are ligand-binding proteins that
have been constructed
based on a lipocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol.
7:8177-8184;
Skerra, A. (2001) J. Biotechnol. 74:257-275). The protein architecture of
lipocalins can include a
beta-barrel having eight antiparallel beta-strands, which supports four loops
at its open end. These
loops form the natural ligand-binding site of the lipocalins, a site which can
be re-engineered ih vitro
by amino acid substitutions to impart novel binding specificities. The amino
acid substitutions can be
made using methods known in the art or described herein, and can include
conservative substitutions
(e.g., substitutions that do not alter binding specificity) or substitutions
that modestly, moderately, or
significantly alter binding specificity.
In one embodiment, screening for compounds which specifically bind to,
stimulate, or inhibit
INTSIG involves producing appropriate cells which express 1NTSIG, either as a
secreted protein or on
the cell membrane. Preferred cells include cells from mammals, yeast, Dt-
osophila, or E. coli. Cells
expressing INTSIG or cell membrane fractions which contain INTSIG are then
contacted with a test
compound and binding, stimulation, or inhibition of activity of either INTSIG
or the compound is
analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example, the
assay may comprise the steps of combining at least one test compound with
1NTSIG, either in solution
or affixed to a solid support, and detecting the binding of IN"hSIG to the
compound. Alternatively, the
assay may detect or measure binding of a test compound in the presence of a
labeled competitor.
Additionally, the assay may be carried out using cell-free preparations,
chemical libraries, or natural
product mixtures, and the test compounds) may be free in solution or affixed
to a solid support.
An assay can be used to assess the ability of a compound to bind to its
natural ligand and/or to
inhibit the binding of its natural ligand to its natural receptors. Examples
of such assays include radio-
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labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S.
Patent No. 6,372,724.
In a related embodiment, one or more amino acid substitutions can be
introduced into a polypeptide
compound (such as a receptor) to improve or alter its ability to bind to its
natural ligands (Matthews,
D.J. and J.A. Wells. (1994) Chem. Biol. 1:25-30). In another related
embodiment, one or more amino
acid substitutions can be introduced into a polypeptide compound (such as a
ligand) to improve or alter
its ability to bind to its natural receptors (Cunningham, B.C. and J.A. Wells
(1991) Proc. Natl. Acad.
Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-
10988).
1NTSIG, fragments of 1NTSIG, or variants of INTSIG may be used to screen for
compounds
that modulate the activity of INTSIG. Such compounds may include agonists,
antagonists, or partial or
to inverse agonists. In one embodiment, an assay is performed under conditions
permissive for INTSIG
activity, wherein INTSIG is combined with at least one test compound, and the
activity of 1NTSIG in
the presence of a test compound is compared with the activity of INTSIG in the
absence of the test
compound. A change in the activity of 7NT'SIG in the presence of the test
compound is indicative of a
compound that modulates the activity of 1NTSIG. Alternatively, a test compound
is combined with an
iyt vitro or cell-free system comprising INTSIG under conditions suitable for
INTSIG activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of
INTSIG rnay do so indirectly and need not come in direct contact with the test
compound. At least
one and up to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding 7NTSIG or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem (ES)
cells. Such techniques are well known in the art and are useful for the
generation of animal models of
human. disease (see, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No.
5,767,337). For example,
mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the
early mouse embryo and
grown in culture. The ES cells are transformed with a vector containing the
gene of interest disrupted
by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi,
M.R. (1989) Science
244:1288-1292). The vector integrates into the corresponding region of the
host genome by
homologous recombination. Alternatively, homologous recombination takes place
using the Cre-loxP
system to knockout a gene of interest in a tissue- or developmental stage-
specific manner (Marth, J.D.
(1996) Clip. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids
Res. 25:4323-4330).
Transformed ES cells are identified and microinjected into mouse cell
blastocysts such as those from
the C57BL/6 mouse strain. The blastocysts are surgically transferred to
pseudopregnant dams, and
the resulting chimeric progeny are genotyped and bred to produce heterozygous
or homozygous
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strains. Transgenic animals thus generated may be tested with potential
therapeutic or toxic agents.
Polynucleotides encoding INTSIG may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding INTSIG can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a region
of a polynucleotide encoding 1NTSIG is injected into animal ES cells, and the
injected sequence
integrates into the animal cell genome. Transformed cells are injected into
blastulae, and the blastulae
are implanted as described above. Transgenic progeny or inbred lines are
studied and treated with
potential pharmaceutical agents to obtain information on treatment of a human
disease. Alternatively,
a mammal inbred to overexpress INTSIG, e.g., by secreting 1NTSIG in its milk,
may also serve as a
convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu.
Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists between
regions of 1NTSIG and intracellular signaling molecules. In addition, examples
of tissues expressing
INTSIG can be found in Table 6 and can also be found in Example XI. In
addition, the expression of
GTPA is closely associated with [From PF-1145 P normal skin, testicular,
endometrial tissues and
diseased lung tissues From PF-1160 brain tumor, dentate nucleus, and smooth
muscle cell tissues, PF-
1162 small intestine and testicular tumor tissues, from PF-1170 P sacral bone
tumor, amygdala and
entorhinal cortex, diseased gallbladder, and small intestine tissues, from PF-
1187 diseased brain tissue,
and normal tissues such as striatum, globus pallidus, posterior putamen,
breast, smooth muscle; spleen,
testicular, and thymus tissues. Therefore, INTSIG appears to play a role in
cell proliferative,
endocrine, autoimmune/inflammatory, neurological, gastrointestinal,
reproductive, developmental, and
vesicle trafficking disorders. In the treatment of disorders associated with
increased INTSIG
expression or activity, it is desirable to decrease the expression or activity
of INTSIG. In the
treatment of disorders associated with decreased INTSIG expression or
activity, it is desirable to
increase the expression or activity of 1NTSIG.
Therefore, in one embodiment, INTSIG or a fragment or derivative thereof may
be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of INTSIG. Examples of such disorders include, but are not limited
to, a cell proliferative
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disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria, polycythemia
vets, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular,
cancers of the adrenal
gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia, gastrointestinal tract,
heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,
prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus; an endocrine disorder such as a
disorder of the
hypothalamus and pituitary resulting from a lesion such as a primary brain
tumor, adenoma, infarction
associated with pregnancy, hypophysectomy, aneurysm, vascular malformation,
thrombosis, infection,
1o immunological disorder, and a complication due to head trauma; a disorder
associated with
hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus,
Kallman's disease,
Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty
sells syndrome, and
dwarfism; a disorder associated with hyperpituitarism including acromegaly,
giantism, and syndrome of
inappropriate antidiuretic hormone secretion (SIADH); a disorder associated
with hypothyroidism
including goiter, myxedema, acute thyroiditis associated with bacterial
infection, subacute thyroiditis
associated with viral infection, autoimmune thyroiditis (Hashimoto's disease),
and cretinism; a disorder
associated with hyperthyroidism including thyrotoxicosis and its various
forms, Grave's disease,
pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and
Plummet's disease; a disorder
associated with hyperparathyroidism including Cone disease (chronic
hypercalemia); a pancreatic
disorder such as Type I or Type II diabetes mellitus and associated
complications; a disorder
associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the
adrenal cortex,
hypertension associated with alkalosis, amyloidosis, hypokalemia, Cushing's
disease, Liddle's
syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and
Addison's disease; a
disorder associated with gonadal steroid hormones such as: in women, abnormal
prolactin production,
infertility, endometriosis, perturbations of the menstrual cycle, polycystic
ovarian disease,
hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea,
galactorrhea, hermaphroditism,
hirsutism and virilization, breast cancer, and, in post-menopausal women,
osteoporosis; and, in men,
Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, a
hypergonadal disorder
associated with a Leydig cell tumor, androgen resistance associated with
absence of androgen
receptors, syndrome of 5 a-reductase, and gynecomastia; an
autoimmune/inflammatory disorder such
as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult
respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis, autoimmune
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hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-
candidiasis-ectodermal
dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's
disease, atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel syndrome,
multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis,
osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and heltninthic
infections, and trauma; a neurological disorder such as epilepsy, ischemic
cerebrovascular disease,
stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's
disease, dementia,
Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral
sclerosis and other motor
neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa,
hereditary ataxias, multiple
sclerosis and other demyelinating diseases, bacterial and viral meningitis,
brain abscess, subdural
empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis
and radiculitis, viral
central nervous system disease, prion diseases including kuru, Creutzfeldt-
Jakob disease, and
Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional
and metabolic diseases
of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system including Down syndrome, cerebral palsy, neuroskeletal
disorders, autonomic nervous
system disorders, cranial nerve disorders, spinal cord diseases, muscular
dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic myopatl>ies, myasthenia gravis,
periodic paralysis, mental
disorders including mood, anxiety, and schizophrenic disorders, seasonal
affective disorder (SAD),
akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,
dystonias, paranoid psychoses,
postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy,
corticobasal degeneration, ,
and familial frontotemporal dementia; a gastrointestinal disorder such as
dysphagia, peptic esophagitis,
esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia,
indigestion, gastritis, gastric
carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema,
abdominal angina, pyrosis,
gastroenteritis, intestinal obstruction, infections of the intestinal tract,
peptic ulcer, cholelithiasis,
cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract
disease, hepatitis,
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hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma,
infectious colitis, ulcerative
colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-
Weiss syndrome, colonic
carcinoma, colonic obstruction, irritable bowel syndrome, short bowel
syndrome, diarrhea, constipation,
gastrointestinal hemorrhage, acquired immunodeficiency syndrome (All~S)
enteropathy, jaundice,
hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis,
hemochromatosis, Wilson's disease,
alphas antitrypsin deficiency, Reye's syndrome, primary sclerosing
cholangitis, liver infarction, portal
vein obstruction and thrombosis, centrilobular necrosis, peliosis hepatis,
hepatic vein thrombosis, veno-
occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy,
intrahepatic cholestasis of
pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and
carcinomas; a
reproductive disorder such as a disorder of prolactin production, infertility,
including tubal disease,
ovulatory defects, endometriosis, a disruption of the estrous cycle, a
disruption of the menstrual cycle,
polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial
or ovarian tumor, a
uterine fibroid, autoimmune disorders, ectopic pregnancy, teratogenesis,
cancer of the breast,
fibrocystic breast disease, galactorrhea, a disruption of spermatogenesis,
abnormal sperm physiology,
cancer of the testis, cancer of the prostate, benign prostatic hyperplasia,
prostatitis, Peyronie's disease,
impotence, carcinoma of the male breast, gynecomastia, hypergonadotropic and
hypogonadotropic
hypogonadism, pseudohermaphroditism, azoospermia, premature ovarian failure,
acrosin deficiency,
delayed puperty, retrograde ejaculation and anejaculation, haemangioblastomas,
cystsphaeochromoeytomas, paraganglioma, cystadenomas of the epididymis, and
endolymphatic sac
tumours; a developmental disorder such as renal tubular acidosis, anemia,
Cushing's syndrome,
achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy,
gonadal dysgenesis,
WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation), Smith-
Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial
dysplasia, hereditary
keratodermas, hereditary neuropathies such as Chareot-Marie-Tooth disease and
neurofibromatosis,
hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea
and cerebral palsy,
spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract,
and sensorineural hearing
loss; and a vesicle trafficking disorder such as cystic fibrosis, glucose-
galactose malabsorption
syndrome, hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper-
and hypoglycemia,
Grave's disease, goiter, Cushing's disease, and Addison's disease,
gastrointestinal disorders including
ulcerative colitis, gastric and duodenal ulcers, other conditions associated
with abnormal vesicle
trafficking, including acquired immunodehciency syndrome (A)DS), allergies
including hay fever,
asthma, and urticaria (hives), autoimmune hemolytic anemia, proliferative
glomerulonephritis,
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inflammatory bowel disease, multiple sclerosis, myasthenia gravis, rheumatoid
and osteoarthritis,
scleroderma, Chediak-Higashi and Sjogren's syndromes, systemic lupus
erythematosus, toxic shock
syndrome, and traumatic tissue damage.
In. another embodiment, a vector capable of expressing INTSIG or a fragment or
derivative
thereof may be administered to a subject to treat or prevent a disorder
associated with decreased
expression or activity of INTSIG including, but not limited to, those
described above.
In a further embodiment, a composition comprising a substantially purified
INTSIG in
conjunction with a suitable pharmaceutical carrier may be administered to a
subject to treat or prevent
a disorder associated with decreased expression or activity of INTSIG
including, but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of INTSIG
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of INTSIG including, but not limited to, those listed above.
In. a further embodiment, an antagonist of INTSIG may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of INTSIG.
Examples of such
disorders include, but are not limited to, those cell proliferative,
endocrine, autoimmune/inflammatory,
neurological, gastrointestinal, reproductive, developmental, and vesicle
trafficking disorders described
above. In one aspect, an antibody which specifically binds INTSIG may be used
directly as an
antagonist or indirectly as a targeting or delivery mechanism for bringing a
pharmaceutical agent to
cells or tissues which express 7NTSIG.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding INTSIG may be administered to a subject to treat or prevent a
disorder associated with
increased expression or activity of INTSIG including, but not limited to,
those described above.
In other embodiments, any protein, agonist, antagonist, antibody,
complementary sequence, or
vector embodiments may be administered in combination with other appropriate
therapeutic agents.
Selection of the appropriate agents for use in combination therapy may be made
by one of ordinary
_ skill in the art, according to conventional pharmaceutical principles. The
combination of therapeutic
agents may act synergistically to effect the treatment or prevention of the
various disorders described
above. Using this approach, one may be able to achieve therapeutic efficacy
with lower dosages of
each agent, thus reducing the potential for adverse side effects.
An antagonist of INTSIG may be produced using methods which are generally
known in the
art. In particular, purified INTSIG may be used to produce antibodies or to
screen libraries of
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pharmaceutical agents to identify those which specifically bind INTSIG.
Antibodies to INTSIG may
also be generated using methods that are well known in the art. Such
antibodies may include, but are
not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies,
Fab fragments, and
fragments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are generally preferred for therapeutic use. Single chain
antibodies (e.g., from
camels or llamas) may be potent enzyme inhibitors and may have advantages in
the design of peptide
mimetics, and in the development of immuno-adsorbents and biosensors
(Muyldermans, S. (2001) J.
Biotechnol. 74:277-302).
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, camels,
1o dromedaries, llamas, humans, and others may be immunized by injection with
1NTSIG or with any
fragment or oligopeptide thereof which has immunogenic properties. Depending
on the host species,
various adjuvants may be used to increase immunological response. Such
adjuvants include, but are
not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface
active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH,
and dinitrophenol. Among
adjuvants used in humans, BCG (bacilli Cahnette-Guerin) and Cozyzzebacterium
parvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
INTSIG have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein. Short stretches
of INTSIG amino acids may be fused with those of another protein, such as KLH,
and antibodies to
the chimeric molecule may be produced.
Monoclonal antibodies to INTSIG may be prepared using any technique which
provides for
the production of antibody molecules by continuous cell lines in culture.
These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-hybridoma
technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al.
(1985) J. Tm_m__unol. Methods
81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030;
Cole, S.P. et al. (1984)
Mol. Cell Biol. 62:109-120).
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used (Morrison, S.L. et
al. (1984) Proc. Natl. Acad.
Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608;
Takeda, S. et al. (1985)
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Nature 314:452-454). Alternatively, techniques described for the production of
single chain antibodies
may be adapted, using methods known in the art, to produce INTSIG-specific
single chain antibodies.
Antibodies with related specificity, but of distinct idiotypic composition,
may be generated by chain
shuffling from random combinatorial immunoglobulin libraries (Burton, D.R.
(1991) Proc. Natl. Acad.
Sci. USA 88:10134-10137).
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening i_m_m__unoglobulin libraries or panels of highly specific
binding reagents as disclosed in
the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-
3837; Winter, G. et al.
(1991) Nature 349:293-299).
Antibody fragments which contain specific binding sites for INTSIG may also be
generated.
For example, such fragments include, but are not limited to, F(ab~2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab~2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity
(Huse, W.D. et al. (1989)
Science 246:1275-1281).
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
1NTSIG and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive
to two non-interfering INTSIG epitopes is generally used, but a competitive
binding assay may also be
employed (Pound, supy-a).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for INTSIG. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of INTSIG-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The Ka
determined for a preparation of polyclonal antibodies, which are heterogeneous
in their affinities for
multiple 7NTSIG epitopes, represents the average affinity, or avidity, of the
antibodies for INTSIG.
The Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a particular
INTSIG epitope, represents a true measure of affinity. High-affinity antibody
preparations with Ka
ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in
which the IN~'SIG-
antibody complex must withstand rigorous manipulations. Low-affinity antibody
preparations with Ka
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ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of INTSIG, preferably in
active form, from the
antibody (Catty, D. (1988) Antibodies Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to determine
the quality and suitability of such preparations for certain downstream
applications. For example, a
polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml, preferably 5-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of INTSIG-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for
antibody quality and usage in various applications, are generally available
(Catty, supra; Coligan et al.,
supra).
In another embodiment of the invention, polynucleotides encoding INTSIG, or
any fragment or
complement thereof, may be used for therapeutic purposes. In one aspect,
modifications of gene
expression can be achieved by designing complementary sequences or antisense
molecules (DNA,
RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of
the gene encoding
INTSIG. Such technology is well known in the art, and antisense
oligonucleotides or larger fragments
can be designed from various locations along the coding or control regions of
sequences encoding
INTSIG (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, Totawa
NJ).
2o In therapeutic use, any gene delivery system suitable for introduction of
the antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein (Slater, J.E. et
al. (1998) J. Allergy Clip. Tm_m__unol. 102:469-475; Scanlon, K.J. et al.
(1995) 9:1288-1296). Antisense
sequences can also be introduced intracellularly through the use of viral
vectors, such as retrovirus and
adeno-associated virus vectors (Miller, A.D. (1990) Blood 76:271; Ausubel et
al., supra; Uckert, W.
and W. Walther (1994) Pharmacol. Ther. 63:323-347). Other gene delivery
mechanisms include
liposome-derived systems, artificial viral envelopes, and other systems known
in the art (Rossi, J.J.
(1995) Br. Med. Bull. 51:217-225; Boado, R.J. et al. (1998) J. Pharm. Sci.
87:1308-1315; Morris,
M.C. et al. (1997) Nucleic Acids Res. 25:2730-2736).
In another embodiment of the invention, polynucleotides encoding INTSIG may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
7i
CA 02458645 2004-02-16
WO 03/031568 PCT/US02/26322
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-
linked inheritance (Ca'vazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
i_mmunodeficiency syndrome associated with an inherited adenosine dearninase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Seience 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
1o cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites (e.g.,
against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D. (1988)
Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA
93:11395-11399), hepatitis
B or C virus (HBV, HCV); fungal parasites, such as Ca~idida albicans and
Paracoccidioides
br-asilierasis; and protozoan parasites such as Plasmodium falcipayunt and
Tfypafaosoma cruzi). In
the case where a genetic deficiency in 1NTSIG expression or regulation causes
disease, the
expression of INTSIG from an appropriate population of transduced cells may
alleviate the clinical
manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
INTSIG are treated by constructing mammalian. expression vectors encoding
INTSIG and introducing
these vectors by mechanical means into 1NTSIG-deficient cells. Mechanical
transfer technologies for
use with cells ifa vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii) ballistic
gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-
mediated gene transfer, and
(v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu.
Rev. Biochem.
62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J.-L. and H. Recipon
(1998) Curr. Opin.
Biotechno1.9:445-450).
Expression vectors that may be effective for the expression of INTSIG include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTI~-HYG (Clontech, Palo Alto CA).
INTSIG
may be expressed using (i) a constitutively active promoter, (e.g., from
cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40 virus, thymidine kinase (TIC), or (3-actin genes),
(ii) an inducible promoter
(e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992)
Proc. Natl. Acad. Sci.
72
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USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi,
F.M.V. and H.M. Blau
(1998) C~rr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen));
the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding INTSIG from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TR.ANSFECTION I~1T, available from Invitrogen) allow one with ordinary skill
in the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of these
standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to INTSIG expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding INTSIG under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R. et
al. (1998) J. Virol. 72:9873-9880). U.5. Patent No. 5,910,434 to Rigg ("Method
for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses
a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4+ T-cells), and the
return of transduced cells to a patient are procedures well known to persons
skilled in the art of gene
therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et
al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al. (1998)
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Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In an embodiment, an adenovirus-based gene therapy delivery system is used to
deliver
polynucleotides encoding INTSIG to cells which have one or more genetic
abnormalities with respect
to the expression of 1N'TSIG. The construction and packaging of adenovirus-
based vectors are well
known to those with ordinary skill in the art. Replication defective
adenovirus vectors have proven to
be versatile for importing genes encoding immunoregulatory proteins into
intact islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for
gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999; Annu.
1o Rev. Nutr. 19:511-544) and Verma, LM. and N. Somia (1997; Nature 18:389:239-
242).
In another embodiment, a herpes-based, gene therapy delivery system is used to
deliver
polynucleotides encoding 7NTSIG to target cells which have one or more genetic
abnormalities with
respect to the expression of INTSIG. The use of herpes simplex virus (HSV)-
based vectors may be
especially valuable for introducing INTSIG to cells of the central nervous
system, for which HSV has
a tropism. The construction and packaging of herpes-based vectors are well
known to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is hereby
incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of
recombinant HSV d92
which consists of a genome containing at least one exogenous gene to be
transferred to a cell under
the control of the appropriate promoter for purposes including human gene
therapy. Also taught by
this patent are the construction and use of recombinant HSV strains deleted
for ICP4, ICP27 and
ICP22. For HSV vectors, see also Goins, W.F. et al. (1999; J. Virol. 73:519-
532) and Xu, H. et al.
(1994; Dev. Biol. 163:152-161). The manipulation of cloned herpesvirus
sequences, the generation of
recombinant virus following the transfection of multiple plasmids containing
different segments of the
large herpesvirus genomes, the growth and propagation of herpesvirus, and the
infection of cells with
herpesvirus are techniques well known to those of ordinary skill in the art.
In another embodiment, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding INTSIG to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
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alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
INTSIG into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
INTSIG-coding RNAs and the synthesis of high levels of INTSIG in vector
transduced cells. While
alphavirus infection is typically associated with cell lysis within a few
days, the ability to establish a
persistent infection in hamster normal kidney cells (BHK-21) with a variant of
Sindbis virus (SIN)
indicates that the lytic replication of alphaviruses can be altered to suit
the needs of the gene therapy
application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host
range of alphaviruses will
allow the introduction of INTSIG into a variety of cell types. The specific
transduction of a subset of
cells in a population may require the sorting of cells prior to transduction.
The methods of
manipulating infectious cDNA clones of alphaviruses, performing alphavirus
cDNA and RNA
transfections, and performing alphavirus infections, are well known to those
with ordinary skill in the
art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions -10
and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can
be achieved using triple helix base-pairing methodology. Triple helix pairing
is useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
been described in the literature (Gee, J.E. et al. (1994) in Huber, B.E. and
B.I. Carr, Molecular and
Tmmunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177). A
complementary
sequence or antisense molecule may also be designed to block translation of
mRNA by preventing the
transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of RNA molecules encoding INTSIG.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
CA 02458645 2004-02-16
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corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes may be prepared by any
method
known in the art for the synthesis of nucleic acid molecules. These include
techniques for chemically
synthesizing oligonucleotides such as solid phase phosphoramidite chemical
synthesis. Alternatively,
RNA molecules may be generated by i~a vitro and iyi vivo transcription of DNA
molecules encoding
INTSIG. Such DNA sequences may be incorporated into a wide variety of vectors
with suitable
RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA
constructs that
synthesize complementary RNA, constitutively or inducibly, can be introduced
into cell lines, cells, or
tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the S' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified
forms of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
2o An additional embodiment of the invention encompasses a method for
screening for a
compound which is effective in altering expression of a polynucleotide
encoding INTSIG. Compounds
which may be effective in altering expression of a specific polynucleotide may
include, but are not
limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming
oligonucleotides,
transcription factors and other polypeptide transcriptional regulators, and
non-macromolecular
chemical entities which are capable of interacting with specific
polynucleotide sequences. Effective
compounds may alter polynucleotide expression by acting as either inhibitors
or promoters of
polynucleotide expression. Thus, in the treatment of disorders associated with
increased INTSIG
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding INTSIG may be therapeutically useful, and in. the treatment of
disorders associated with
decreased INTSIG expression or activity, a compound which specifically
promotes expression of the
polynucleotide encoding 1NTSIG may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
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altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding INTSIG is exposed to at least one test compound thus
obtained. 'The sample
may comprise, for example, an intact or permeabilized cell, or an in vitro
cell-free or reconstituted
biochemical system. Alterations in the expression of a polynucleotide encoding
INTSIG are assayed
by any method commonly known in the art. Typically, the expression of a
specific nucleotide is
detected by hybridization with a probe having a nucleotide sequence
complementary to the sequence
of the polynucleotide encoding INTSIG. The amount of hybridization may be
quantified, thus forming
the basis for a comparison of the expression of the polynucleotide both with
and without exposure to
one or more test compounds. Detection of a change in the expression of a
polynucleotide exposed to
a test compound indicates that the test compound is effective in altering the
expression of the
polynucleotide. A screen for a compound effective in altering expression of a
specific polynucleotide
can be carried out, for example, using a Schizosacchat-otnyees pombe gene
expression system
(Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000)
Nucleic Acids Res.
28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000)
Biochem. Biophys. Res.
Commun. 268:8-13). A particular embodiment of the present invention involves
screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide nucleic
acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide sequence
(Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al.
(2000) U.S. Patent No.
6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ~x vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art (Goldman, C.K. et al. (1997)
Nat. Biotechnol. 15:462-
466).
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
CA 02458645 2004-02-16
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monkeys.
An additional embodiment of the invention relates to the administration of a
composition which
generally comprises an active ingredient formulated with a pharmaceutically
acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and
proteins. Various
formulations are commonly known and are thoroughly discussed in the latest
edition of Remin~ton's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of 1NTSIG,
antibodies to INTSIG, and mimetics, agonists, antagonists, or inhibitors of
INTSIG.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary, intrathecal,
intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of fast-
acting formulations is well-known in the art. In the case of macromolecules
(e.g. larger peptides and
proteins), recent developments in the held of pulmonary delivery via the
alveolar region of the lung
have enabled the practical delivery of drugs such as insulin. to blood
circulation (see, e.g., Patton, J.S.
et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of
administration without
needle injection, and obviates the need for potentially toxic penetration
enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising INTSIG or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, INTSIG or a fragment thereof may be joined to a
short cationic N-
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs, monkeys,
or pigs. An animal model may also be used to determine the appropriate
concentration range and
'7s
CA 02458645 2004-02-16
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route of administration. Such information can then be used to determine useful
doses and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
1NTSIG or fragments thereof, antibodies of 1NTSIG, and agonists, antagonists
or inhibitors of INTSIG,
which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity
may be determined
by standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDso (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDso/EDso ratio. Compositions
which exhibit large
1o therapeutic indices are preferred. The data obtained from cell culture
assays and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDso
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and response
to therapy. Long-acting compositions may be administered every 3 to 4 days,
every week, or
biweekly depending on the half life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1,ug to 100,000 ,ug, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind INTSIG may be used
for the
diagnosis of disorders characterized by expression of INTSIG, or in assays to
monitor patients being
treated with INTSIG or agonists, antagonists, or inhibitors of INTSIG.
Antibodies useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic
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assays for INTSIG include methods which utilize the antibody and a label to
detect INTSIG in human
body fluids or in extracts of cells or tissues. The antibodies may be used
with or without modification,
and may be labeled by covalent or non-covalent attachment of a reporter
molecule. A wide variety of
reporter molecules, several of which are described above, are known in the art
and may be used.
A variety of protocols for measuring INTSIG, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
INTSIG expression. Normal
or standard values for I1VTSIG expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibodies to INTSIG
under conditions suitable for complex formation. The amount of standard
complex formation may be
quantitated by various methods, such as photometric means. Quantities of
INTSIG expressed in
subject, control, and disease samples from biopsied tissues are compared with
the standard values.
Deviation between standard~and subject values establishes the parameters for
diagnosing disease.
In another embodiment of the invention, polynucleotides encoding INTSIG may be
used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotides, complementary
RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and
quantify gene
expression in biopsied tissues in which expression of INTSIG may be correlated
with disease. The
diagnostic assay may be used to determine absence, presence, and excess
expression of 1NTSIG, and
to monitor regulation of INTSIG levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotides,
including genomic sequences, encoding 1NTSIG or closely related molecules may
be used to identify
nucleic acid sequences which encode INTSIG. The specificity of the probe,
whether it is made from
a highly specific region, e.g., the 5'regulatory region, or from a less
specific region, e.g., a conserved
motif, and the stringency of the hybridization or amplification will determine
whether the probe
identifies only naturally occurring sequences encoding INTSIG, allelic
variants, or related sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the INTSIG encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:46-90 or from
genomic sequences including promoters, enhancers, and introns of the INTSIG
gene.
Means for producing specific hybridization probes for polynucleotides encoding
INTSIG
include the cloning of polynucleotides encoding INTSIG or INTSIG derivatives
into vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitf-o by means of the addition of the
appropriate RNA
so
CA 02458645 2004-02-16
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polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotides encoding INTSIG may be used for the diagnosis of disorders
associated with
expression of INTSIG. Examples of such disorders include, but are not limited
to, a cell proliferative
disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria, polycythemia
vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular,
cancers of the adrenal
gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia, gastrointestinal tract,
heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,
prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus; an endocrine disorder such as a
disorder of the
hypothalamus and pituitary resulting from a lesion such as a primary brain
tumor, adenoma, infarction
associated with pregnancy, hypophysectomy, aneurysm, vascular malformation,
thrombosis, infection,
immunological disorder, and a complication due to head trauma; a disorder
associated with
hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus,
Kallinan's disease,
Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty
sella syndrome, and
dwarfism; a disorder associated with hyperpituitarism including acromegaly,
giantism, and syndrome of
inappropriate antidiuretic hormone secretion (SIADH); a disorder associated
with hypothyroidism
including goiter, myxedema, acute thyroiditis associated with bacterial
infection, subacute thyroiditis
associated with viral infection, autoimmune thyroiditis (Hashimoto's disease),
and cretinism; a disorder
associated with hyperthyroidism including thyrotoxicosis and its various
forms, Grave's disease,
pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and
Plummer's disease; a disorder
associated with hyperparathyroidism including Cone disease (chronic
hypercalemia); a pancreatic
disorder such as Type I or Type lI diabetes mellitus and associated
complications; a disorder
associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the
adrenal cortex,
hypertension associated with alkalosis, amyloidosis, hypokalemia, Cushing's
disease, Liddle's
syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and
Addison's disease; a
disorder associated with gonadal steroid hormones such as: in women, abnormal
prolactin production,
infertility, endometriosis, perturbations of the menstrual cycle, polycystic
ovarian disease,
hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea,
galactorrhea, hermaphroditism,
hirsutism and virilization, breast cancer, and, in post-menopausal women,
osteoporosis; and, in men,
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Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, a
hypergonadal disorder
associated with a Leydig cell tumor, androgen resistance associated with
absence of androgen
receptors, syndrome of 5 a-reductase, and gynecomastia; an
autoimmune/inflammatory disorder such
as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult
respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis, autoimmune
hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-
candidiasis-ectodermal
dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's
disease, atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins,
eryrhroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel syndrome,
multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis,
osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and helminthic
infections, and trauma; a neurological disorder such as epilepsy, ischemic
cerebrovascular disease,
stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's
disease, dementia,
Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral
sclerosis and other motor
neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa,
hereditary ataxias, multiple
sclerosis and other demyelinating diseases, bacterial and viral meningitis,
brain abscess, subdural
empyema, epidural abscess, suppurative intxacranial thrombophlebitis, myelitis
and radiculitis, viral
central nervous system disease, priors diseases including kuru, Creutzfeldt-
Jakob disease, and
Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional
and metabolic diseases
of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system including Down syndrome, cerebral palsy, neuroskeletal
disorders, autonomic nervous
system disorders, cranial nerve disorders, spinal cord diseases, muscular
dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis,
periodic paralysis, mental
disorders including mood, anxiety, and schizophrenic disorders, seasonal
affective disorder (SAD),
akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,
dystonias, paranoid psychoses,
postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy,
corticobasal degeneration,
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and familial frontotemporal dementia; a gastrointestinal disorder such as
dysphagia, peptic esophagitis,
esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia,
indigestion, gastritis, gastric
carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema,
abdominal angina, pyrosis,
gastroenteritis, intestinal obstruction, infections of the intestinal tract,
peptic ulcer, cholelithiasis,
cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract
disease, hepatitis,
hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma,
infectious colitis, ulcerative
colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-
Weiss syndrome, colonic
carcinoma, colonic obstruction, irritable bowel syndrome, short bowel
syndrome, diarrhea, constipation,
gastrointestinal hemorrhage, acquired immunodeficiency syndrome (AIDS)
enteropathy, jaundice,
hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis,
hemochromatosis, Wilson's disease,
alphas-antitrypsin deficiency, Reye's syndrome, primary sclerosing
cholangitis, liver infarction, portal
vein obstruction and thrombosis, centrilobular necrosis, peliosis hepatis,
hepatic vein thrombosis, veno-
occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy,
intrahepatic cholestasis of
pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and
carcinomas; a
reproductive disorder such as a disorder of prolactin production, infertility,
including tubas disease,
ovulatory defects, endometriosis, a disruption of the estrous cycle, a
disruption of the menstrual cycle,
polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial
or ovarian tumor, a
uterine fibroid, autoimmune disorders, ectopic pregnancy, teratogenesis,
cancer of the breast,
fibrocystic breast disease, galactorrhea, a disruption of spermatogenesis,
abnormal sperm physiology,
cancer of the testis, cancer of the prostate, benign prostatic hyperplasia,
prostatitis, Peyronie's disease,
impotence, carcinoma of the male breast, gynecomastia, hypergonadotropic and
hypogonadotropic
hypogonadism, pseudohermaphroditism, azoospernua, premature ovarian failure,
acrosin deficiency,
delayed puperty, retrograde ejaculation and anejaculation, haemangioblastomas,
cystsphaeochromocytomas, paraganglioma, cystadenomas of the epididymis, and
endolymphatic sac
tumours; a developmental disorder such as renal tubular acidosis, anemia,
CS.ishing's syndrome,
achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy,
gonadal dysgenesis,
WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation), Smith-
Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial
dysplasia, hereditary
keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and
neurofibromatosis,
hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea
and cerebral palsy,
spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract,
and sensorineural hearing
loss; and a vesicle trafficking disorder such as cystic fibrosis, glucose-
galactose malabsorption
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syndrome, hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper-
and hypoglycemia,
Grave's disease, goiter, Cushing's disease, and Addison's disease,
gastrointestinal disorders including
ulcerative colitis, gastric and duodenal ulcers, other conditions associated
with abnormal vesicle
trafficking, including acquired immunodeficiency syndrome (AIDS), allergies
including hay fever,
asthma, and urticaria (hives), autoimmune hemolytic anemia, proliferative
glomerulonephritis,
inflammatory bowel disease, multiple sclerosis, myasthenia gravis, rheumatoid
and osteoarthritis,
scleroderma, Chediak-Higashi and Sjogren's syndromes, systemic lupus
erythematosus, toxic shock
syndrome, and traumatic tissue damage. Polynucleotides encoding 1NTSIG may be
used in Southern
or northern analysis, dot blot, or other membrane-based technologies; in PCR
technologies; in dipstick,
pin, and multiformat ELISA-like assays; and in xnicroarrays utilizing fluids
or tissues from patients to
detect altered INTSIG expression. Such qualitative or quantitative methods are
well known in the art.
In a particular aspect, polynucleotides encoding 1NTSIG may be used in assays
that detect the
presence of associated disorders, particularly those mentioned above.
Polynucleotides complementary
to sequences encoding INTSIG may be labeled by standard methods and added to a
fluid or tissue
sample from a patient under conditions suitable for the formation of
hybridization complexes. After a
suitable incubation period, the sample is washed and the signal is quantified
and compared with a
standard value. If the amount of signal in the patient sample is significantly
altered in comparison to a
control sample then the presence of altered levels of polynucleotides encoding
7NTSIG in the sample
indicates the presence of the associated disorder. Such assays may also be
used to evaluate the
efficacy of a particular therapeutic treatment regimen in animal studies, in
clinical trials, or to monitor
the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of
INTSIG, a normal or standard profile for expression is established. This may
be accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof, encoding INTSIG, under conditions suitable
for hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from normal
subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
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patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development
of the disease, or may provide a means for detecting the disease prior to the
appearance of actual
clinical symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier, thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding INTSIG
may involve the use of PCR. These oligomers may be chemically synthesized,
generated
enzymatically, or produced in vitf-o. Oligomers will preferably contain a
fragment of a polynucleotide
encoding IN'TSIG, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
INTSIG, and will be employed under optimized conditions for identification of
a specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from polynucleotides
encoding 1NTSIG
may be used to detect single nucleotide polymorphisms (SNPs). SNPs are
substitutions, insertions and
deletions that are a frequent cause of inherited or acquired genetic disease
in humans. Methods of
SNP detection include, but are not limited to, single-stranded conformation
polymorphism (SSCP) and
fluorescent SSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived
from polynucleotides
encoding INTSIG are used to amplify DNA using the polymerase chain reaction
(PCR). The DNA'
may be derived, for example, from diseased or normal tissue, biopsy samples,
bodily fluids, and the
like. SNPs in the DNA cause differences in the secondary and tertiary
structures of PCR products in
single-stranded form, and these differences are detectable using gel
electrophoresis in non-denaturing
gels. In fSCCP, the oligonucleotide primers are fluorescently labeled, which
allows detection of the
amplimers in high-throughput equipment such as DNA sequencing machines.
Additionally, sequence
database analysis methods, termed in silico SNP (isSNP), are capable of
identifying polymorphisms by
comparing the sequence of individual overlapping DNA fragments which assemble
into a common
consensus sequence. These computer-based methods filter out sequence
variations due to laboratory
preparation of DNA and sequencing errors using statistical models and
automated analyses of DNA
sequence chromatograms. In the alternative, SNPs may be detected and
characterized by mass
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spectrometry using, for example, the high throughput MASSARRAY system
(Sequenom, Inc., San
Diego CA).
SNPs may be used to study the genetic basis of human disease. For example, at
least 16
common SNPs have been associated with non-insulin-dependent diabetes mellitus.
SNPs are also
useful for examining differences in disease outcomes in monogenic disorders,
such as cystic fibrosis,
sickle cell anemia, or chronic granulomatous disease. For example, variants in
the manuose-binding
lectin, MBL2, have been shown to be correlated with deleterious pulmonary
outcomes in cystic
fibrosis. SNPs also have utility in pharmacogenomics, the identification of
genetic variants that
influence a patient's response to a drug, such as life-threatening toxicity.
For example, a variation in
N-acetyl transferase is associated with a high incidence of peripheral
neuropathy in response to the
anti-tuberculosis drug isoniazid, while a variation in the core promoter of
the ALOXS gene results in
diminished clinical response to treatment with an anti-asthma drug that
targets the 5-lipoxygenase
pathway. Analysis of the distribution of SNPs in different populations is
useful for investigating
genetic drift, mutation, recombination, and selection, as well as for tracing
the origins of populations
and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
Kwok, P.-Y. and Z. Gu
(1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr. Opin.
Neurobiol. 11:637-641).
Methods which may also be used to quantify the expression of INTSIG include
radiolabeling
or biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves (Melby, P.C. et al. (1993) J. Tm_m__unol. Methods 159:235-244;
Duplaa, C. et al. (1993)
Anal. Biochem. 212:229-236). The speed of quantitation of multiple samples
maybe accelerated by
running the assay in a high-throughput format where the oligomer or
polynucleotide of interest is
presented in various dilutions and a spectrophotometric or colorimetric
response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotides described herein may be used as elements on a microarray. The
microarray can be
used in transcript imaging techniques which monitor the relative expression
levels of large numbers of
genes simultaneously as described below. The microarray may also be used to
identify genetic
variants, mutations, and polymorphisms. This information may be used to
determine gene function, to
understand the genetic basis of a disorder, to diagnose a disorder, to monitor
progression/regression of
disease as a function of gene expression, and to develop and monitor the
activities of therapeutic
agents in the treatment of disease. In particular, this information may be
used to develop a
pharmacogenomic profile of a patient in order to select the most appropriate
and effective treatment
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regimen for that patient. For example, therapeutic agents which are highly
effective and display the
fewest side effects may be selected for a patient based on his/her
pharmacogenomic profile.
In another embodiment, INTSIG, fragments of INTSIG, or antibodies specific for
1NTSIG
may be used as elements on a microarray. The microarray may be used to monitor
or measure
protein-protein interactions, drug-target interactions, and gene expression
profiles, as described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S.
Patent No. 5,840,484;
hereby expressly incorporated by reference herein). Thus a transcript image
may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression iya vivo, as in the
ease of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also be used in conjunction with iii vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000)
Toxicol. Lett. 112-113:467-471). If a test compound has a signature similar to
that of a compound
with known toxicity, it is likely to share those toxic properties. These
fingerprints or signatures are
most useful and refined when they contain expression information from a large
number of genes and
gene families. Ideally, a genome-wide measurement of expression provides the
highest quality
signature. Even genes whose expression is not altered by any tested compounds
are important as
well, as the levels of expression of these genes are used to normalize the
rest of the expression data.
The normalization procedure is useful for comparison of expression data after
treatment with different
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compounds. While the assignment of gene function to elements of a toxicant
signature aids in
interpretation of toxicity mechanisms, knowledge of gene function is not
necessary for the statistical
matching of signatures which leads to prediction of toxicity (see, for
example, Press Release 00-02
from the National Institute of Environmental Health Sciences, released
February 29, 2000, available at
http://www.niehs.nih.gov/oc/news/toxchip.htm). Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In an embodiment, the toxicity of a test compound can be assessed by treating
a biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
treated biological sample are hybridized with one or more probes specific to
the polynucleotides of the
present invention, so that transcript levels corresponding to the
polynucleotides of the present invention
may be quantified. The transcript levels in the treated biological sample are
compared with levels in
an untreated biological sample. Differences in the transcript levels between
the two samples are
indicative of a toxic response caused by the test compound in the treated
sample.
Another embodiment relates to the use of the polypeptides disclosed herein to
analyze the
proteome of a tissue or cell type. The term proteome refers to the global
pattern of protein expression
in a particular tissue or cell type. Each protein component of a proteome can
be subjected individually
to further analysis. Proteome expression patterns, or profiles, are analyzed
by quantifying the number
of expressed proteins and their relative abundance under given conditions and
at a given time. A
profile of a cell's proteome may thus be generated by separating and analyzing
the polypeptides of a
particular tissue or cell type. In one embodiment, the separation is achieved
using two-dimensional gel
electrophoresis, in which proteins from a sample are separated by isoelectric
focusing in the first
dimension, and then according to molecular weight by sodium dodecyl sulfate
slab gel electrophoresis
in the second dimension (Steiner and Anderson, supra). The proteins are
visualized in the gel as
discrete and uniquely positioned spots, typically by staining the gel with an
agent such as Coomassie
Blue or silver or fluorescent stains. The optical density of each protein spot
is generally proportional to
the level of the protein in the sample. The optical densities of equivalently
positioned protein spots
from different samples, for example, from biological samples either treated or
untreated with a test
compound or therapeutic agent, are compared to identify any changes in protein
spot density related to
the treatment. The proteins in the spots are partially sequenced using, for
example, standard methods
employing chemical or enzymatic cleavage followed by mass spectrometry. The
identity of the protein
in a spot may be determined by comparing its partial sequence, preferably of
at least 5 contiguous
amino acid residues, to the polypeptide sequences of interest. In some cases,
further sequence data
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may be obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for 1NTSIG
to quantify
the levels of INTSIG expression. In one embodiment, the antibodies are used as
elements on a
microarray, and protein expression levels are quantified by exposing the
microarray to the sample and
detecting the levels of protein bound to each array element (Lueking, A. et
al. (1999) Anal. Biochem.
270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection
may be performed by
a variety of methods known in the art, for example, by reacting the proteins
in the sample with a thiol-
or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at each
array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to rapid
degradation of mRNA, so proteomic profiling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in, an
untreated biological sample.
A difference in the amount of protein between the two samples is indicative of
a toxic response to the
test compound in the treated sample. Individual proteins are identified by
sequencing the amino acid
residues of the individual proteins and comparing these partial sequences to
the polypeptides of the
present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared
with the amount in an untreated biological sample. A difference in the amount
of protein between the
two samples is indicative of a toxic response to the test compound in the
treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the art
(Brennan,
T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc.
Natl. Acad. Sci. USA
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93:10614-10619; Baldeschweiler et al. (1995) PCT application WO95/251116;
Shalon, D. et al. (1995)
PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci.
USA 94:2150-2155;
Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662). Various types of
microarrays are well known
and thoroughly described in Schena, M., ed. (1999; DNA Microarrays: A
Practical Approach, Oxford
University Press, London).
In another embodiment of the invention, nucleic acid sequences encoding INTSIG
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances,
noncoding sequences may
be preferable over coding sequences. For example, conservation of a coding
sequence among
members of a multi-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
region of a chromosome, or to artificial chromosome constructions, e.g., human
artiftcial chromosomes
(HACs), yeast artificial chromosomes (PACs), bacterial artificial chromosomes
(BACs), bacterial P1
constructions, or single chromosome cDNA libraries (Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-
355; Price, C.M. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet.
7:149-154). Once
mapped, the nucleic acid sequences may be used to develop genetic linkage
maps, for example, which
correlate the inheritance of a disease state with the inheritance of a
particular chromosome region or
restriction fragment length polymorphism (RFLP) (Larder, E.S. and D. Botstein
(1986) Proc. Natl.
Acad. Sci. USA 83:7353-7357).
Fluorescent i~c situ hybridization (FISH) may be correlated with other
physical and genetic
map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples
of genetic map data
can be found in various scientific journals or at the Online Mendelian
Inheritance in Man (OMIM)
World Wide Web site. Correlation between the location of the gene encoding
INTSIG on a physical
map and a specific disorder, or a predisposition to a specific disorder, may
help define the region of
DNA associated with that disorder and thus may further positional cloning
efforts.
Iu situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to 11q22-23, any
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sequences mapping to that area may represent associated or regulatory genes
for further investigation
(Gatti, R.A. et al. (1988) Nature 336:577-580). The nucleotide sequence of the
instant invention may
also be used to detect differences in the chromosomal location due to
translocation, inversion, etc.,
among normal, carrier, or affected individuals.
In another embodiment of the invention, INTSIG, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between INTSIG and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest (Geysers, et al.
(1984) PCT application
W084/03564). In this method, large numbers of different small test compounds
are synthesized on a
solid substrate. The test compounds are reacted with INTSIG, or fragments
thereof, and washed.
Bound IN'pSIG is then detected by methods well known in the art. Purified
INTSIG can also be
coated directly onto plates for use in the aforementioned drug screening
techniques. Alternatively,
non-neutralizing antibodies can be used to capture the peptide and immobilize
it on a solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding 7NTSIG specifically compete with a test compound
for binding 1NTSIG.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with INTSIG.
In additional embodiments, the nucleotide sequences which encode INTSIG may be
used in
any molecular biologgy techniques that have yet to be developed, pxovided the
new techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are, therefore,
to be construed as merely illustrative, and not limitative of the remainder of
the disclosure in any way
whatsoever.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
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The disclosures of all patents, applications, and publications mentioned above
and below,
including U.S. Ser. No. 60/313,245, U.S. Ser. No. 60/314,751, U.S. Ser. No.
60/316,752, U.S. Ser.
No. 60/316,847, U.S. Ser. No. 60/322,188, U.S. Ser. No. 60/326,390, U.S. Ser.
No. 60/328,952, U.S.
Ser. No. 60/345,468, and U.S. Ser. No. 60/372,499, are hereby expressly
incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 3. Some
tissues were
homogenized and lysed in guanidinium isothiocyanate, while others were
homogenized and lysed in
phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), a
monophasic solution of
phenol and guanidine isothiocyanate. The resulting lysates were centrifuged
over CsCl cushions or
extracted with chloroform. RNA was precipitated from the lysates with either
isopropanol or sodium
acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was
isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles
(QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively,
RNA was isolated directly from tissue lysates using other RNA isolation kits,
e.g., the
POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the
UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen),
using the
recommended procedures or similar methods known in the art (Ausubel et al.,
supf-a, ch. 5). Reverse
transcription was initiated using oligo d(T) or random primers. Synthetic
oligonucleotide adapters were
ligated to double stranded cDNA, and the cDNA was digested with the
appropriate restriction enzyme
or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using
SEPHACRYL
S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham
Biosciences) or preparative agarose gel electrophoresis. cDNAs were ligated
into compatible
restriction enzyme sites of the polylinker of a suitable plasmid, e.g.,
PBLUESCRIPT plasmid
(Stratagene), PSPORT1 plasmid (Invitrogen), PCDNA2.1 plasmid (Invitrogen,
Carlsbad CA), PBK-
CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid
(Stratagene),
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pIGEN (Ineyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pINCY
(Incyte Genomics),
or derivatives thereof. Recombinant plasmids were transformed into competent
E. coli cells including
XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX
DH10B
from Invitrogen.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
ih vivo
excision using the UNI2AP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
were resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at 4
°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation such
as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200
thermal cycler
(MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Biosciences or supplied in ABI sequencing
kits such as the
ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Amersham
Biosciences);
the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard
ABI protocols and base calling software; or other sequence analysis systems
known in the art.
Reading frames within the cDNA sequences were identified using standard
methods (Ausubel et al.,
supra, ch. 7). Some of the cDNA sequences were selected for extension using
the techniques
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disclosed in Example Vla.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Flomo
Sapiens, Rattus ytofvegicus, Mus musculus, Caehorhabditis elegans, Sacchar-
omyces cet~evisiae,
Schizosaccharomyces pombe, and Candida albica~ts (Incyte Genomics, Palo Alto
CA); hidden
l0 Markov model (HMM)-based protein family databases such as PFAM, INCY~ and
TIGRFAM (Haft,
D.H. et al. (2001) Nucleic Acids Res. 29:41-43); and HIVllVI-based protein
domain databases such as
SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864;
Letunic, I. et al. (2002)
Nucleic Acids Res. 30:242-244). (HMM is a probabilistic approach which
analyzes consensus
primary structures of gene families; see, for example, Eddy, S.R. (1996) Curr.
Opin. Struct. Biol.
6:361-365.) The queries were performed using programs based on BLAST, FASTA,
BLM'S, and
F~~IMER. The Incyte cDNA sequences were assembled to produce full length
polynucleotide
sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences,
stretched
sequences, or Genscan-predicted coding sequences (see Examples IV and V) were
used to extend
Incyte cDNA assemblages to full length. Assembly was performed using programs
based on Phred,
Phrap, and Consed, and cDNA assemblages were screened for open reading frames
using programs
based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences
were translated
to derive the corresponding full length polypeptide sequences. Alternatively,
a polypeptide may begin
at any of the methionine residues of the full length translated polypeptide.
Full length polypeptide
sequences were subsequently analyzed by querying against databases such as the
GenBank protein
databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO,
PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such
as PFAM,
INCY, and TIGRFAM; anal HMM-based protein domain databases such as SMART. Full
length
polynucleotide sequences are also analyzed using MACDNASIS PRO software
(MiraiBio, Alameda
CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence
alignments
are generated using default parameters specified by the CLUSTAL algorithm as
incorporated into the
MEGALIGN multisequence alignment program (DNASTAR), which also calculates the
percent
identity between aligned sequences.
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Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and threshold
parameters. The first column of Table 7 shows the tools, programs, and
algorithms used, the second
column provides brief descriptions thereof, the third column presents
appropriate references, all of
which are incorporated by reference herein in their entirety, and the fourth
column presents, where
applicable, the scores, probability values, and other parameters used to
evaluate the strength of a
match between two sequences (the higher the score or the lower the probability
value, the greater the
identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide and
polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:46-90. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies are described in Table 4, column 2.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative intracellular signaling molecules were initially identified by
running the Genscan gene
identification program against public genomic sequence databases (e.g., gbpri
and gbhtg). Genscan is
a general-purpose gene identification program which analyzes genomic DNA
sequences from a
variety of organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94;
Burge, C. and S. Karlin
(1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates
predicted exons to form an
assembled cDNA sequence extending from a methionine to a stop codon. The
output of Genscan is a
FASTA database of polynucleotide and polypeptide sequences. The maximum range
of sequence for
Genscan to analyze at once was set to 30 kb. To determine which of these
Genscan predicted cDNA
sequences encode intracellular signaling molecules, the encoded polypeptides
were analyzed by
querying against PFAM models for intracellular signaling molecules. Potential
intracellular signaling
molecules were also identified by homology to Incyte cDNA sequences that had
been annotated as
intracellular signaling molecules. These selected Genscan-predicted sequences
were then compared
by BLAST analysis to the genpept and gbpri public databases. Where necessary,
the Genscan-
predicted sequences were then edited by comparison to the top BLAST hit from
genpept to correct
errors in the sequence predicted by Genscan, such as extra or omitted exons.
BLAST analysis was
also used to find any Incyte cDNA or public cDNA coverage of the Genscan-
predicted sequences,
thus providing evidence for transcription. When Incyte cDNA coverage was
available, this
information was used to correct or confirm the Genscan predicted sequence.
Full length
polynucleotide sequences were obtained by assembling Genscan-predicted coding
sequences with
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Iucyte cDNA sequences and/or public cDNA sequences using the assembly process
described in
Example la. Alternatively, full length polynucleotide sequences were derived
entirely from edited or
unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
~~Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
DI were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
~~Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenB~ protein homolog.
Insertions or deletions
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may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for homologous
genomic sequences from the public human genome databases. Partial DNA
sequences were
therefore "stretched" or extended by the addition of homologous genomic
sequences. The resultant
stretched sequences were examined to determine whether it contained a complete
gene.
VI. Chromosomal Mapping of INTSIG Encoding Polynucleotides
The sequences which were used to assemble SEQ ID NO:46-90 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
1o SEQ ID N0:46-90 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
15 of all sequences of that cluster, including its particular SEQ ll~ NO:, to
that map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
20 humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances
are based on genetic markers mapped by Genethon which provide boundaries for
radiation hybrid
markers whose sequences were included in each of the clusters. Human genome
maps and other
resources available to the public, such as the NCBI "GeneMap'99" World Wide
Web site
(http://www.ncbi.nlm.nih.govlgenemap~, can be employed to determine if
previously identified disease
25 genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound (Sambrook, supf-a, ch.
7; Ausubel et al., supf-a,
3o ch.4).
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
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much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
search can be modified to determine whether any particular match is
categorized as exact or similar.
The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
1o calculated as follows: the BLAST score is multiplied by the percent
nucleotide identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotides encoding 1NTSIG are analyzed with respect to
the tissue
sources from which they were derived. For example, some full length sequences
are assembled, at
least in part, with overlapping Ineyte cDNA sequences (see Example DI). Each
cDNA sequence is
derived from a eDNA library constructed from a human tissue. Each human tissue
is classified into
one of the following organ/tissue categories: cardiovascular system;
connective tissue; digestive
system; embryonic structures; endocrine system; exocrine glands; genitalia,
female; genitalia, male;
germ cells; heroic and immune system; liver; musculoskeletal system; nervous
system; pancreas;
respiratory system; sense organs; skin; stomatognathic system;
unclassified/mixed; or urinary tract.
The number of libraries in each category is counted and divided by the total
number of libraries across
all categories. Similarly, each human tissue is classified into one of the
following disease%ondition
categories: cancer, cell line, developmental, inflammation, neurological,
trauma, cardiovascular, pooled,
and other, and the number of libraries in each category is counted and divided
by the total number of
libraries across all categories. The resulting percentages reflect the tissue-
and disease-specific
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expression of cDNA encoding INTSIG. cDNA sequences and cDNA library/tissue
information are
found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
VIII. Extension of INTSIG Encoding Polynucleotides
Full length polynucleotides are produced by extension of an appropriate
fragment of the full
length molecule using oligonucleotide primers designed from this fragment. One
primer was
synthesized to initiate 5' extension of the known fragment, and the other
primer was synthesized to
initiate 3' extension of the known fragment. The initial primers were designed
using OLIGO 4.06
software (National Biosciences), or another appropriate program, to be about
22 to 30 nucleotides in
length, to have a GC content of about 50% or more, and to anneal to the target
sequence at
temperatures of about 68 °C to about 72 °C. Any stretch of
nucleotides which would result in hairpin
structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High h.delity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH~)zSO4,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE
enzyme
(Invitrogen), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer pair
PCIA and PCIB: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step
3: 60°C, 1 min; Step 4: 68°C, 2
min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min;
Step 7: storage at 4 °C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94 °C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68 °C, 5 min; Step 7: storage at 4 °C.
The concentration of DNA in each well was determined by dispensing 100 ~,1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~Cl of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 /.c1 to 10 ,u1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in e~endi_ng the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
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digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to relegation into pUC 18 vector (Amersham
Biosciences). For shotgun
sequencing, the digested nucleotides were separated on low concentration (0.6
to 0.8%) agarose gels,
fragments were excised, and agar digested with Agar ACE (Promega). Extended
clones were
religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector
(Amersham
Biosciences), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site overhangs, and
transfected into competent E. coli cells. Transformed cells were selected on
antibiotic-containing
media, and individual colonies were picked and cultured overnight at 37
°C in 384-well plates in LB/2x
curb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Biosciences) and Pfu DNA polymerase (Stratagene) with the following
parameters: Step
1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1
min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and
4 repeated 29 times; Step 6: 72 °C, 5 min; Step 7: storage at 4
°C. DNA was quantified by
PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries
were reamplified using the same conditions as described above. Samples were
diluted with 20%
dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing primers
and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotides are verified using the above
procedure or are used
to obtain 5'regulatory sequences using the above procedure along with
oligonucleotides designed for
such extension, and an appropriate genomic library.
IX. Identification of Single Nucleotide Polymorphisms in INTSIG Encoding
Polynucleotides
Common DNA sequence variants known as single nucleotide polymorphisms (SNPs)
were
identified in SEQ 1D NO:46-90 using the LIFESEQ database (Incyte Genomics).
Sequences from the
same gene were clustered together and assembled as described in Example III,
allowing the
identification of all sequence variants in the gene. An algorithm consisting
of a series of filters was
used to distinguish SNPs from other sequence variants. Preliminary filters
removed the majority of
basecall errors by requiring a minim__um Phred quality score of 15, anal
removed sequence alignment
errors and errors resulting from improper trimm;r,g of vector sequences,
chimeras, and splice variants.
An automated procedure of advanced chromosome analysis analysed the original
chromatogram files
in the vicinity of the putative SNP. Clone error filters used statistically
generated algorithms to identify
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errors introduced during laboratory processing, such as those caused by,
reverse transcriptase,
polymerase, or somatic mutation. Clustering error filters used statistically
generated algorithms to
identify errors resulting from clustering of close homologs or pseudogenes, or
due to contamination by
non-human sequences. A final set of filters removed duplicates and SNPs found
in immunoglobulins
S or T-cell receptors.
Certain SNPs were selected for further characterization by mass spectrometry
using the high
throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at
the SNP sites in
four different human populations. The Caucasian population comprised 92
individuals (46 male, 46
female), including 83 from Utah, four French, three Venezualan, and two Amish
individuals. The
African population comprised 194 individuals (97 male, 97 female), all African
Americans. The
Hispanic population comprised 324 individuals (162 male, 162 female), all
Mexican Hispanic. The
Asian population comprised 126 individuals (64 male, 62 female) with a
reported parental breakdown
of 43 % Chinese, 31 % Japanese, 13 % Korean, 5 % Vietnamese, and 8 % other
Asian. Allele
frequencies were first analyzed in the Caucasian population; in some cases
those SNPs which showed
no allelic variance in this population were not further tested in the other
three populations.
X. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ D7 NO:46-90 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ,uCi of
[y-3aP] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide
kinase (DuPont NEN,
Boston MA). The labeled oligonucleotides are substantially purified using a
SEPHADEX G-25
superfine size exclusion dextran bead column. (Amersham Biosciences). An
aliquot containing 10'
counts per minute of the labeled probe is used in a typical membrane-based
hybridization analysis of
human genomic DNA digested with one of the following endonucleases: Ase I, Bgl
1I, Eco RI, Pst I,
Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytxan Plus, Schleicher & Schuell, Durham NHS. Hybridization is
carried out for 16
hours at 40 °C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
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compared.
XI. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing; see, e.g.,
Baldeschweiler et al., supra),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena, M., ed.
(1999) DNA Microarrays: A Practical Approach, Oxford University Press,
London). Suggested
substrates include silicon, silica, glass slides, glass chips, and silicon
wafers. Alternatively, a procedure
analogous to a dot or slot blot may also be used to arrange and link elements
to the surface of a
substrate using thermal, UV, chemical, or mechanical bonding procedures. A
typical array may be
produced using available methods and machines well known to those of ordinary
skill in the art and
may contain any appropriate number of elements (Schena, M. et al. (1995)
Science 270:467-470;
Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson
(1998) Nat. Biotechnol.
16:27-31).
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
2o After hybridization, nonhybridized nucleotides from the biological sample
are removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described
in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~,1 oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/~.1 RNase inhibitor, 500 ~.M dATP, 500 ~.M dGTP, 500
p,M dTTP, 40 p.M
dCTP, 40 ~,M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse
transcription
reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with
GEMBRIGHT kits
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(Incyte). Specific control poly(A)+ RNAs are synthesized by ih vitro
transcription from non-coding
yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction
sample (one with Cy3 and
another with Cy5 labeling) is treated with 2.5 m1 of O.SM sodium hydroxide and
incubated for 20
minutes at 85° C to the stop the reaction and degrade the RNA. Samples
are purified using two
successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories,
Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen {1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and resuspended
in 14 ~,l 5X SSC/0.2% SDS.
Microarray Pr~aration
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5 p,g.
Amplified array elements are then purified using SEPHACRYh-400 (Amersham
Biosciences).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 p1 of the array
element DNA, at an average
concentration of 100 ng/~Cl, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslivker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2%
SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 p1 of sample mixture consisting of 0.2 ~.g
each of Cy3 and
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Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of 140
~.l of 5X SSC in a corner of the chamber. The chamber containing the arrays is
incubated for about
6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in
a first wash buffer (1X SSC, 0.1 %
SDS), three times for 10 minutes each at 45° C in a second wash buffer
(0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 mn for excitation of Cy3 and at 632 llm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that location
to be correlated with a weight ratio of hybridizing species of 1:100,000. When
two samples from
different sources (e.g., representing test and control cells), each labeled
with a different fluorophore,
are hybridized to a single array for the purpose of identifying genes that are
differentially expressed,
the calibration is done by labeling samples of the calibrating cDNA with the
two fluorophores and
adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an 7BM-
compatible PC
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computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping emission
spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
to obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
Array elements
that exhibited at least about a two-fold change in expression, a signal-to-
background ratio of at least
2.5, and an element spot size of at least 40% were identified as
differentially expressed using the
GEMTOOLS program (Incyte Genomics).
Expression
For example, SEQ ID N0:54 was differentially expressed in human peripheral
blood
mononuclear cells (PBMCs) treated with 10 ng/ml interleukin 4 (IL-4). Human
PBMCs can be
classified into discrete cellular populations representing the major cellular
components of the immune
system. PBMCs contain about 52% lymphocytes (12 % B lymphocytes, 40% T
lymphocytes {25%
CD4+ and 15% CD8+}), 20% NK cells, 25% monocytes, and 3% various cells that
include dendritic
cells and progenitor cells. The proportions, as well as the biology of these
cellular components tend to
vary slightly between healthy individuals, depending on factors such as age,
gender, past medical
history, and genetic background.
1L-4 is a pleiotropic cytokine produced by activated T cells, mast cells, and
basophils. It was
initially identified as a B cell differentiation factor (BCDF) and a B cell
stimulatory factor (BSF1).
Subsequent to the molecular cloning and expression of both human and mouse IL-
4, numerous other
functions have been ascribed to B cells and other hematopoietic and non-
hematopoietic cells including
endothelial cells, etc. IL-4 exhibits anti-tumor effects both iyi vivo and in
vitro. Recently, IL,-4 was
identified as an important regulator for the CD4+ subset (Thl-like vs. Th2-
like) development. The
biological effects of IL-4 are mediated by the binding of IL,-4 to specific
cell surface receptors. The
functional high-affinity receptor for IL-4 consists of a ligand-binding
subunit (IL-4 R) and a second
subunit (b chain) that can modulate the ligand binding aff'mity of the
receptor complex. In certain cell
types, the gamma chain of the 1L-2 receptor complex is a functional b chain of
the IL-4 receptor
complex.
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In this experiment, PBMCs were collected from the blood of 6 healthy volunteer
donors using
standard gradient separation. The PBMCs from each donor were placed in culture
for 2 hours in the
presence or absence of recombinant 1L-4. Treated PBMCs and untreated control
PBMCs from the
different donors were pooled according to their respective treatment. The
expression of SEQ ll~
N0:54 was significantly decreased by at least two-fold in the PBMCs treated
with 1L-4.
Also, SEQ ll7 N0:66 showed differential expression in inflammatory responses
as determined
by microarray analysis. Compared to untreated peripheral blood mononuclear
cells (PBMCs) (12% B
lymphocytes, 40% T lymphocytes, 20% NK cells, 25% monocytes, and 3% various
cells that include
dendritic and progenitor cells), the expression of SEQ ID NO:66 was increased
by at least 2 fold in
1o PBMCs treated with either Interleukin-1 beta (1Z=1 (3), Interleukin-6 (7I,-
6), or TNF-oc. 1L-1 [3 is a
prototypical pro-inflammatory cytokine; II,-6 is a multifunctional protein
important in immune
responses; and TNF-cc is a pleotropic cytokine which mediates inflammatory
responses through signal
transduction pathways. Therefore, SEQ ID N0:66 is useful as a diagnostic
marker for inflammatory
responses.
Further, SEQ ID N0:88 showed increased expression in peripheral blood
mononuclear cells
(PBMCs) treated with 25 microM prednisone versus untreated cells as determined
by microarray
analysis. PBMCs from the blood of 6 healthy volunteer donors were incubated
for 24 hours in the
presence of graded doses of prednisone dissolved in ethanol. In addition,
matching PBMCs were
treated for the same duration with matching doses of ethanol to monitor the
possible effects of the
vehicle alone. Treated PBMCs were compared to matching untreated PBMCs
maintained in culture
for the same duration. Further, SEQ m N0:88 showed increased expression in
PBMCs treated with
Staphlococcal endotoxin B (SEB) versus untreated cells. PBMCs from 7 healthy
volunteer donors
were stimulated i~ vitro with SEB for 72 hours. The SEB-treated PBMCs from
each donor were
compared to PBMCs from the same donor, kept in culture for 24 hours in the
absence of SEB.
Therefore, in various embodiments, SEQ ID N0:54, SEQ >D N0:66, and SEQ ID
NO:88 can be used
for one or more of the following: i) monitoring treatment of immune disorders
and related diseases and
conditions, ii) diagnostic assays. for immune disorders and related diseases
and conditions, and iii)
developing therapeutics and/or other treatments for immune disorders and
related diseases and
conditions.
Colon cancer develops through a multi-step.process in which pre-malignant
colonocytes
undergo a relatively defined sequence of events leading to tumor formation.
Factors that contribute to
the process of tumor progression and malignant transformation include
genetics, mutations, and
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selection. The expression of SEQ ID N0:54 was significantly decreased by at
least two-fold in
various experiments involving colon adenocarcinoma tissue compared to
uninvolved tissue from the
same donor. Further, SEQ D7 NO:90 showed differential expression associated
with colon cancer, as
determined by microarray analysis. Gene expression profiles from the following
matched samples
were compared: normal colon and colon tumor tissue from a 56-year-old female
diagnosed with poorly
differentiated metastatic adenocarcinoma of possible ovarian origin and a
clinical history of recurrent
cecal mass (Huntsman Cancer Institute, Salt Lake City, UT); normal and tumor
samples from a 58-
year-old female diagnosed with mucinous adenocarcinoma (Huntsman Cancer
Institute, Salt Lake
City, U°T); normal and tumor samples from an 83-year-old female
diagnosed with colon cancer
(Huntsman Cancer Institute, Salt Lake City, UT); and normal and tumor samples
from a 64-year-old
female diagnosed with moderately differentiated colon adenocarcinoma (Huntsman
Cancer Institute,
Salt Lake City, UT). The expression of SEQ )D N0:90 was downregulated by at
least.two-fold in
tumor tissues as compared to normal colon tissue. Therefore, in various
embodiments, SEQ ID N0:54
and SEQ ~ NO:90 can be used for one or more of the following: i) monitoring
treatment of colon
cancer, ii) diagnostic assays for colon cancer, and iii) developing
therapeutics and/or other treatments
for colon cancer.
As with most tumors, prostate cancer develops through a multistage progression
ultimately
resulting in an aggressive tumor phenotype. 'The initial step in tumor
progression involves the hyper-
proliferation of normal luminal and/or basal epithelial cells. Androgen
responsive cells become
hyperplastic and evolve into early-stage tumors. Although early-stage tumors
are often androgen
sensitive and respond to androgen ablation, a population of androgen
independent cells evolve from the
hyperplastic population. These cells represent a more advanced form of
prostate tumor that may
become invasive and potentially become metastatic to the bone, brain, or lung.
The expression of SEQ
117 N0:55 was differentially expressed in DU145 cells, a line of prostate
carcinoma cells isolated from
a metastatic site in the brain of a 69-year old male with widespread
metastatic prostate carcinoma, as
compared to PrEC cells, a primary prostate epithelial cell lice isolated from
a normal donor. DU145
has no detectable sensitivity to hormones; forms colonies in semi-solid
medium; is only weakly positive
for acid phosphatase; and cells are negative for prostate specific antigen
(PSA). The expression of
SEQ ID N0:55 was increased by at least two-fold in prostate tumor cells.
Additional experiments conducted to compare gene expression profiles yielded
differential
expression of SEQ ID NO:55. PrEC/3 is a primary prostate epithelial cell line
isolated from a normal
donor. Prostate carcinoma cell lines DU145 and PC3 (metastatic prostate
adenocarcinoma) were
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compared to PrEC/3 cells. Under these conditions, the expression of SEQ ~
NO:55 was increased
by at least two-fold in the tumor cell lines.
In a similar experiment, the gene expression profiles of prostate carcinoma
cell lines DU145
and LNCaP grown under optimal conditions were compared to those of PrEC/3s
grown under
restrictive conditions. The expression of SEQ m N0:55 was decreased by at
least two-fold in the
tumor cell lines.
4.,. s i
Also, SEQ m N0:69 and SEQ m N0:70 showed differential expression in prostate a
adenocarcinoma cells versus normal prostate epithelial cells as determined by
microarray analysis.
The prostate adenocarcinoma cell line was isolated from a metastatic site in
the bone of a 62 year old
1o male with grade IV prostate adenocarcinoma. The expression of SEQ m N0:69
and SEQ D7 N0:70
were increased by at least two fold in a prostate carcinoma cell line relative
to normal prostate
epithelial cells. Therefore, in various embodiments, SEQ )D N0:55 and SEQ >D
NO:69-70 can be
used for one or more of the following: i) monitoring treatment of prostate
cancer, ii) diagnostic assays
for prostate cancer, and iii) developing therapeutics and/or other treatments
for prostate cancer.
Lung cancers are divided into four histopathologically distinct groups. Three
groups
(squamous cell carcinoma, adenocarcinoma, and large cell carcinoma) are
classified as non-small cell
lung cancers (NSCLCs). The fourth group of cancer is referred to as small cell
lung cancer (SCLC). ,
Collectively, NSCLCs account for approximately 70% of cases while SCLCs
account for
approximately 18% of all cases. Pair comparisons were performed in which
normal lung tissue and
lung tumor tissue from the same donor were examined. Two squamous cell
carcinomas were
compared to same-donor normal lung tissue, yielding an increase in the
expression of SEQ >D NO:55
by at least two-fold in all cases. Further, SEQ )D NO:86 showed differential
expression in lung tumor
tissue as determined by microarray analysis. Lung cancer is the leading cause
of cancer death for
men and the second leading cause of cancer death for women in the U.S. Lung
cancers are divided
into four histopathologically distinct groups. Three groups (squamous cell
carcinoma, adenocarcinoma
and large cell carcinoma) are classified as non-small cell lung cancers, while
the fourth group is
classified as small cell lung cancer. Non-small cell lung cancers account for
about 70% of lung
cancer cases. Pair comparisons of normal and tumor tissue were performed with
matched tissue
samples from a 73-year old male patient exhibiting squamous cell carcinoma.
Results showed that
expression of SEQ )D NO:86 in the tumor tissue is decreased by at least two-
fold. Therefore, in
various embodiments, SEQ ~ N0:55 and SEQ )D N0:86 can be used for one or more
of the
following: i) monitoring treatment of lung cancer, u) diagnostic assays for
lung cancer, and iii)
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developing therapeutics and/or other treatments for lung cancer.
In another example, as determined by microarray analysis, SEQ ID NO:65 showed
differential
expression when comparing cells from a metastatic breast tumor cell line
versus primary breast
epithelial cells and non-malignant mammary epithelial cells. The metastatic
breast tumor cell line,
MDA-mb-X31, was derived from the pleural effusion of a 51-year-old female with
metastatic breast
carcinoma; the primary breast epithelial cell line, HMEC was isolated from a
normal donor; and the
non-malignant mammary epithelial cell line, MCF10A, was isolated from a 36-
year-old female with
fibrocystic breast disease. All cell cultures were propagated in a defined
media, according to the
supplier's recommendations and grown to 70-80% confluence prior to RNA
isolation. The microarray
experiments showed that the expression of SEQ ID N0:65 was increased by at
least two fold in the
metastatic breast tumor cell line relative to the primary breast epithelial
cells and the non-malignant
mammary epithelial cells. Therefore, in various embodiments, SEQ ID NO:65 can
be used for one or
more of the following: r) monitoring treatment of breast cancer, ii)
diagnostic assays for breast cancer,
and iii) developing therapeutics and/or other treatments for breast cancer.
SEQ ID N0:65 also showed differential expression in preadipocytes versus
differentiated
adipocytes as determined by microarray analysis. The primary function of
adipose tissue is the ability
to store and release fat during periods of feeding and fasting. Understanding
how the various
molecules regulate adiposity in physiological and pathological situations is
important for developing
diagnostic and therapeutic tools for human obesity. Adipose tissue is also one
of the primary target
tissues for insulin, and adipogenesis and insulin resistance are linked in non-
insulin dependent diabetes
mellitus. Cytologically, the conversion of a preadipocytes into mature
adipocytes is characterized by
deposition of fat droplets around the nuclei. The conversion process in vivo
can be induced by
thiazolidinediones and other peroxisome proliferator-activated receptor gamma
(PPARy) agonists
(Adams et al. (1997) J. Clip. Invest. 100:3149-3153) which are new classes of
anti-diabetic agents
which improve insulin sensitivity and reduce plasma glucose and blood pressure
in patients with type II
diabetes. Some PPARy agents have been proven to induce human adipocyte
differentiation. For
these assays, human primary preadipocytes were isolated from adipose tissue of
a 36 year old healthy
female with body mass index 27.7 and a 40 year old healthy female with a body
mass index of 32.47.
The preadipocytes were cultured and induced to differentiate into adipocytes
by culturing them in a
medium containing PPARy agonist and human insulin. The microarray experiments
showed that the
expression of SEQ ID NO:65 was decreased by at least two fold in preadipocytes
treated with
PPARy agonists and insulin relative to untreated preadipocytes. Therefore, SEQ
ll~ NO:65 is useful
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as a diagnostic marker or as a potential therapeutic target for obesity and
diabetes. Therefore, in
various embodiments, SEQ ll~ N0:65 can be used for one or more of the
following: i) monitoring
treatment of obesity and diabetes, ii) diagnostic assays for obesity and
diabetes, and iii) developing
therapeutics and/or other treatments for obesity and diabetes.
In another example, SEQ ID N0:71 showed differential expression in human
ovarian
adenocarcenomic tissue as compared to normal ovarian tissue from the same
donor. Ovarian cancer
is the leading cause of death from a gynecologic cancer. The majority of
ovarian cancers are derived
from epithelial cells, and 70% of patients with epithelial ovarian cancers
present with late-stage
disease. As a result the loingterm survival rates for this disease are very
low. Identification of early
stage markers for ovarian cancer would significantly increase the survival
rate. The molecular events
that lead to ovarian cancer are poorly understood. Some of the known
aberrations include mutation of
p53 and microsatellite instability. Since gene expression patterns likely vary
when normal ovary is
compared to ovarian tumors we have examined gene expression inm these tissues
to identify possible
markers for ovarian cancer. The expression of SEQ ID N0:71 was significantly
increased by at least
two-fold in ovarian tissue as compared to normal tissue. Therefore, in various
embodiments, SEQ ID
N0:71 can be used for one or more of the following: i) monitoring treatment of
ovarian cancer, ii)
diagnostic assays for ovarian cancer, and iii) developing therapeutics and/or
other treatments for
ovarlan Cancer.
The effects upon liver metabolism and hormone clearance mechanisms are
important to
understand the pharmacodynamics of a drug. For example, the human C3A cell
line is a clonal
derivative of HepG2/C3 (hepatoma cell line, isolated from a 15-year-old male
with liver tumor), which
was selected for strong contact inhibition of growth. The use of a clonal
population enhances the
reproducibility of the cells. C3A cells have many characteristics of primary
human hepatocytes in
culture: i) expression of insulin receptor and insulin-like growth factor II
receptor; ii) secretion of a
high ratio of serum albumin compared with a-fetoprotein; iii) conversion of
ammonia to urea and
glutamine; iv) metabolism of aromatic amino acids; and v) proliferation in
glucose-free and insulin-
free medium. The C3A cell line is now well established as an in vitro model of
the mature human liver
(Mickelson et al. (1995) Hepatology 22:866-875; Nagendra et al. (1997) Am. J.
Physiol. 272:G408-
G416). In another example, SEQ ll~ N0:75, SEQ m N0:77-81 and SEQ ID NO:84
showed
increased expression in C3A cells treated with a beclomethasone,
betamethasone, budesonide,
medroxyprogesterone, prednisone, and progesterone, versus untreated C3A Bells,
as determined by
microarray analysis. Therefore, in various embodiments, SEQ ID N0:75, SEQ ID
N0:77-81 and
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SEQ ID N0:84 can be used for one or more of the following: i) monitoring
treatment of liver and
immune disorders and related diseases and conditions, ii) diagnostic assays
for liver and immune
disorders and related diseases and conditions, and iii) developing
therapeutics and/or other treatments
for liver and immune disorders and related diseases and conditions.
XII. Complementary Polynucleotides
Sequences complementary to the INTSIG-encoding sequences, or any parts
thereof, are used
to detect, decrease, or inhibit expression of naturally occurring INTSIG.
Although use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of INTSIG.
To inhibit transcription, a complementary oligonucleotide is designed from the
most unique 5' sequence
and used to prevent promoter binding to the coding sequence. To inhibit
translation, a complementary
oligonucleotide is designed to prevent ribosomal binding to the INTSIG-
encoding transcript.
XIII. Expression of INTSIG
Expression and purification of INTSIG is achieved using bacterial or virus-
based expression
systems. For expression of INTSIG in bacteria, cDNA is subcloned into an
appropriate vector
containing an. antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
tfp-lac (tac) hybrid
promoter and the TS or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express INTSIG upon induction with isopropyl
beta-D-
thiogalactopyranoside (IPTG). Expression of INTSIG in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Autogr-aphica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding INTSIG by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA trauscription. Recombinant
baculovirus is used to
infect Spodopter~a, fi-ugipef-da (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus (Engelhard, E.K. et al.
(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.
Gene Ther. 7:1937-
1945).
In most expression systems, INTSIG is synthesized as a fusion protein with,
e.g., glutathione
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S-trausferase (GST) or a peptide epitope tag, such as FLAG or 6-His,
permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
enzyme from Scltistosoma japo~zicum, enables the purification of fusion
proteins on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Biosciences).
Following purification, the GST moiety can be proteolytically cleaved from
1NTSIG at specifically
engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity
purification using
commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman
Kodak). 6-His, a
stretch of six consecutive histidine residues, enables purification on metal-
chelate resins (QIAGEN).
Methods for protein expression and purification are discussed in Ausubel et
al. (supra, ch. 10 and 16).
Purified INTSIG obtained by these methods can be used directly in the assays
shown in Examples
XV1I, XVIII, and XVIII, where applicable.
XIV. Functional Assays
1NTSIG function is assessed by expressing the sequences encoding 1NTSIG at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid
(Invitrogen), both of
which contain the cytomegalovirus promoter. S-10 tcg of recombinant vector are
transiently
transfected into a human cell line, for example, an endothelial or
hematopoietic cell line, using either
liposome formulations or electroporation. 1-2 ~g of an additional plasmid
containing sequences
encoding a marker protein are co-transfected. Expression of a marker protein
provides a means to
distinguish trausfected cells from nontransfected cells and is a reliable
predictor of cDNA expression
from the recombinant vector. Marker proteins of choice include, e.g., Green
Fluorescent Protein
(GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser
optics-based technique, is used to identify transfected cells expressing GFP
or CD64-GFP and to
evaluate the apoptotic state of the cells and other cellular properties. FCM
detects and quantifies the
uptake of fluorescent molecules that diagnose events preceding or coincident
with cell death. These
events include changes in nuclear DNA content as measured by staining of DNA
with propidium
iodide; changes in cell size and granularity as measured by forward light
scatter and 90 degree side
light scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine
uptake; alterations in expression of cell surface and intracellular proteins
as measured by reactivity
with specific antibodies; and alterations in plasma membrane composition as
measured by the binding
of fluorescein-conjugated Annexin V protein to the cell surface. Methods in
flow cytometry are
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discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York NY).
The influence of 1NTSIG on gene expression can be assessed using highly
purified populations
of cells transfected with sequences encoding INTSIG and either CD64 or CD64-
GFP. CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind to
conserved regions of human
immunoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success
NY). mRNA can be purified from the cells using methods well known by those of
skill in the art.
Expression of mRNA encoding INTSIG and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
1o XV. Production of INTSIG Specific Antibodies
INTSIG substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using
standard protocols.
Alternatively, the 1NTSIG amino acid sequence is analyzed using LASERGENE
software
15 (DNASTAR) to determine regions of high i_m_n_iunogenicity, and a
corresponding oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art (Ausubel et al., supf-a, ch. 11).
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
20 peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled
to KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with
the oligopeptide-KLH
complex in. complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-INTSIG
activity by, for example, binding the peptide or INTSIG to a substrate,
blocking with 1 % BSA, reacting
25 with rabbit antisera, washing, and reacting with radio-iodinated goat anti-
rabbit IgG.
XVI. Purification of Naturally Occurring INTSIG Using Specific Antibodies
Naturally occurring or recombinant 1NTSIG is substantially purified by
immunoaffmity
chromatography using antibodies specific for INTSIG. An immunoaffinity column
is constructed by
covalently coupling anti-INTSIG antibody to an activated chromatographic
resin, such as
30 CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the
resin is blocked and
washed according to the manufacturer's instructions.
Media containing 1NTSIG are passed over the immunoaffinity column, and the
column is
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washed under conditions that allow the preferential absorbance of INTSIG
(e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/INTSIG binding (e.g., a buffer of pH 2 to pH 3, or a high
concentration of a chaotrope, such
as urea or thiocyanate ion), and 1NTSIG is collected.
XVII. Identification of Molecules Which Interact with INTSIG
INTSIG, or biologically active fragments thereof, are labeled with lzsl Bolton-
Hunter reagent
(Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539). Candidate
molecules previously
arrayed in the wells of a multi-well plate are incubated with the labeled
IN'I'SIG, washed, and any
wells with labeled INTSIG complex are assayed. Data obtained using different
concentrations of
INTSIG are used to calculate values for the number, affinity, and association
of INTSIG with the
candidate molecules.
Alternatively, molecules interacting with INTSIG are analyzed using the yeast
two-hybrid
system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
INTSIG may also be used in the PATHCALLING process (CuraGen Corp., New Haven
CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) IJ.S.
Patent No. 6,057,101).
XVIII. Demonstration of INTSIG Activity
IN'rSIG activity is associated with its ability to form protein-protein
complexes and is
measured by its ability to regulate growth characteristics of NIH3T3 mouse
fibroblast cells. A cDNA
encoding INTSIG is subcloned into an appropriate eukaryotic expression vector.
This vector is
txansfected into NIH3T3 cells using methods known in the art. Transfected
cells are compared with
non-transfected cells for the following quantifiable properties: growth in
culture to high density,
reduced attachment of cells to the substrate, altered cell morphology, and
ability to induce tumors
when injected into immunodeficient mice. The activity of INTSIG is
proportional to the extent of
increased growth or frequency of altered cell morphology in NI)=I3T3 cells
transfected with INTSIG.
Alternatively, INTSIG activity is measured by binding of IN'TSIG to
radiolabeled formin
polypeptides containing the proline-rich region that specifically binds to SH3
containing proteins (Char,
D.C. et al. (1996) EMBO J. 15:1045-1054). Samples of INTSIG are run on SDS-
PAGE gels, and
transferred onto nitrocellulose by electroblotting. The blots are blocked for
1 hr at room temperature
in TBST (137 mM NaCl, 2.7 mM KCl, 25 mM Tris (pH 8.0) and 0.1% Tween-20)
containing non-fat
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dry milk. Blots are then incubated with TBST containing the radioactive formin
polypeptide for 4 hrs
to overnight. After washing the blots four times with TBST, the blots are
exposed to autoradiographic
film. Radioactivity is quantitated by cutting out the radioactive spots and
counting them in a
radioisotope counter. The amount of radioactivity recovered is proportional to
the activity of INTSIG
in the assay.
Alternatively, PDE activity of INTSIG is measured by monitoring the conversion
of a cyclic
nucleotide (either cAMP or cGMP) to its nucleotide monophosphate. The use of
tritium-containing
substrates such as 3H-cAMP and 3H-cGMP, and 5' nucleotidase from snake venom,
allows the PDE
reaction to be followed using a scintillation counter. cAMP-specific PDE
activity of INTSIG is
assayed by measuring the conversion of 3H-cAMP to 3H-adenosine in the presence
of INTSIG and 5'
nucleotidase. A one-step assay is run using a 100 ~.1 reaction containing 50
mM Tris-HCl pH 7.5, 10
mM MgClz, 0.1 unit 5' nucleotidase (from Crotalus atr~ox venom), 0.0062-0.1
~.M 3H-cAMP, and
various concentrations of cAMP (0.0062-3 mM). The reaction is started by the
addition of 25 ~.1 of
diluted enzyme supernatant. Reactions are run directly in mini Poly-Q
scintillation vials (Beckman
Instruments, Fullerton CA). Assays are incubated at 37 °C for a time
period that would give less than
15% cAMP hydrolysis to avoid non-linearity associated with product inhibition.
The reaction is
stopped by the addition of 1 ml of Dowex (Dow Chemical, Midland MI) AGlxB (C1
form) resin (1:3
slurry). Three ml of scintillation fluid are added, and the vials are mixed.
The resin in the vials is
allowed to settle for one hour before counting. Soluble radioactivity
associated with 3H-adenosine is
quantitated using a beta scintillation counter. The amount of radioactivity
recovered is proportional to
the cAMP-specific PDE activity of INTSIG in the reaction. For inhibitor or
agonist studies, reactions
are carried out under the conditions described above, with the addition of 1%
DMSO, 50 nM cAMP,
and various concentrations of the inhibitor or agonist. Control reactions are
carried out with all
reagents except for the enzyme aliquot.
In an alternative assay, cGMP-specific PDE activity of 1NTSIG is assayed by
measuring the
conversion of 3H-cGMP to 3H-guanosine in the presence of INTSIG and 5'
nucleotidase. A one-step
assay is run using a 100 ~.l reaction containing 50 mM Tris-HCl pH 7.5, 10 mM
MgC>2, 0.1 unit 5'
nucleotidase (from Cr~otalus atf-ox venom), and 0.0064-2.0 ~.M 3H-cGMP. The
reaction is started by
the addition of 25 ~.l of diluted enzyme supernatant. Reactions are run
directly in mini Poly-Q
scintillation vials (Beckman Instruments). Assays are incubated at 37
°C for a time period that would
yield less than 15% cGMP hydrolysis in order to avoid non-linearity associated
with product inhibition.
The reaction is stopped by the addition of 1 ml of Dowex (Dow Chemical,
Midland MI) AGlx8 (C1
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form) resin ( 1:3 slurry). Three ml of scintillation fluid are added, and the
vials are mixed. The resin in
the vials is allowed to settle for one hour before counting. Soluble
radioactivity associated with 3H-
guanosine is quantitated using a beta scintillation counter. The amount of
radioactivity recovered is
proportional to the cGMP-specific PDE activity of 7NTSIG in the reaction. For
inhibitor or agonist
studies, reactions are carried out under the conditions described above, with
the addition of 1%
DMSO, 50 nM cGMP, and various concentrations of the inhibitor or agonist.
Control reactions are
carried out with all reagents except for the enzyme aliquot.
Alternatively, IN'TSIG protein kinase activity is measured by quantifying the
phosphorylation
of an appropriate substrate in the presence of gamma-labeled 32P-ATP. 1NTSIG
is incubated with the
1o substrate, 32P-ATP, and an appropriate kinase buffer. The 32P incorporated
into the product is
separated from free 3aP-ATP by electrophoresis, and the incorporated 32P is
quantified using a beta
radioisotope counter. The amount of incorporated 32P is proportional to the
protein kinase activity of
1NTSIG in the assay. A determination of the speciEc amino acid residue
phosphorylated by protein
kinase activity is made by phosphoamino acid analysis of the hydrolyzed
protein.
Alternatively, an assay for INTSIG protein phosphatase activity measures the
hydrolysis of
para-nitrophenyl phosphate (PNPP). INTSIG is incubated together with PNPP in
HEPES buffer pH
7.5, in the presence of 0.1% (3-mercaptoethanol at 37 °C for 60 min.
The reaction is stopped by the
addition of 6 ml of 10 N NaOH, and the increase in light absorbance of the
reaction mixture at 410 nm
resulting from the hydrolysis of PNPP is measured using a spectrophotometer.
The increase in light
absorbance is proportional to the activity of INTSIG in the assay (Diamond,
R.H. et al. (1994) Mol.
Cell Biol. 14:3752-3762).
Alternatively, adenylyl cyclase activity of INTSIG is demonstrated by the
ability to convert
ATP to cAMP (Mittal, C.K. (1986) Meth. Enzymol. 132:422-428). In this assay
INTSIG is incubated
with the substrate [a-32P]ATP, following which the excess substrate is
separated from the product
cyclic [32P] AMP. INTSIG activity is determined in 12 x 75 mm disposable
culture tubes containing 5
~tl of 0.6 M Tris-HCl, pH 7.5, 5 ~.l of 0.2 M MgCla, 5 ~,l of 150 mM creatine
phosphate containing 3
units of creatine phosphokinase, 5 ~,l of 4.0 mM 1-methyl-3-isobutylxanthine,
5 ~.1 of 20 mM cAMP, 5
p1 20 mM dithiothreitol, 5 ~.l of 10 mM ATP, 10 ~.l [a-32P]ATP (2-4 x 106
cpm), and water in a total
volume of 100 ~,1. The reaction mixture is prewarmed to 30 °C. The
reaction is initiated by adding
IN'TSIG to the prewarmed reaction mixture. After 10-15 minutes of incubation
at 30 °C, the reaction
is terminated by adding 25 p1 of 30% ice-cold trichloroacetic acid (TCA). Zero-
time incubations and
reactions incubated in the absence of INTSIG are used as negative controls.
Products are separated
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by ion exchange chromatography, and cyclic [32P] AMP is quantified using a (3-
radioisotope counter.
The INTSIG activity is proportional to the amount of cyclic [32P) AMP formed
in the reaction.
An alternative assay measures 1NTSIG-mediated G-protein signaling activity by
monitoring
the mobilization of Ca2+ as au indicator of the signal transduction pathway
stimulation. (See, e.g.,
Grynkiewicz, G. et al. (1985) J. Biol. Chem. 260:3440; McColl, S. et al.
(1993) J. Tm_m__unol.
150:4550-4555; and Aussel supra). The assay requires preloading neutrophils or
T cells with a
fluorescent dye such as FURA-2 or BCECF (Universal Imaging Corp, Westchester
PA) whose
emission characteristics are altered by Caa+ binding. When the cells are
exposed to one or more
activating stimuli artificially (e.g., anti-CD3 antibody ligation of the T
cell receptor) or physiologically
(e.g., by allogeneic stimulation), Ca2+ flux takes place. This flux can be
observed and quantified by
assaying the cells in a fluorometer or fluorescent activated cell sorter.
Measurements of Ca2+ flux are
compared between cells in their normal state and those transfected with
1NTSIG. Increased Caa+
mobilization attributable to increased 1NTSIG concentration is proportional to
INTSIG activity.
Alternatively, GTP-binding activity of INTSIG is determined in an assay that
measures the
binding of 1NTSIG to [a-32P]-labeled GTP. Purified INTSIG is first blotted
onto filters and rinsed in a
suitable buffer. The filters are then incubated in buffer containing
radiolabeled [a-32P]-GTP. The
filters are washed in buffer to remove unbound GTP and counted in a
radioisotope counter. Non-
specific binding is determined in an assay that contains a 100-fold excess of
unlabeled GTP. The
amount of specific binding is proportional to the activity of INTSIG.
Alternatively, GTPase activity of INTSIG is determined in au assay that
measures the
conversion of [a-32P)-GTP to [a-32P]-GDP. INTSIG is incubated with [a-32P]-GTP
in buffer for au
appropriate period of time, and the reaction is terminated by heating or acid
precipitation followed by
centrifugation. An aliquot of the supernatant is subjected to polyacrylamide
gel electrophoresis
(PAGE) to separate GDP and GTP together with unlabeled standards. The GDP spot
is cut out and
counted in a radioisotope counter. The amount of radioactivity recovered in
GDP is proportional to the
GTPase activity of INTSIG.
Alternatively,1NTSIG activity is measured by quantifying the amount of a non-
hydrolyzable
GTP analogue, GTFyS, bound over a 10 minute incubation period. Varying amounts
of INTSIG are
incubated at 30 °C in 50 mM Tris buffer, pH 7.5, containing 1 mM
dithiothreitol, 1 mM EDTA and 1
~.M [ssS]GTFyS. Samples are passed through nitrocellulose filters and washed
twice with a buffer
consisting of 50 mM Tris-HCl, pH 7.8, 1 mM NaN3, 10 mM MgClz, 1 mM EDTA, 0.5
mM
dithiothreitol, 0.01 mM PMSF, and 200 mM NaCl. The filter-bound counts are
measured by liquid
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scintillation to quantify the amount of bound [35S]GTFyS. 1NTSIG activity may
also be measured as
the amount of GTP hydrolysed over a 10 minute incubation period at 37
°C. INTSIG is incubated in
SOmM Tris-HCl buffer, pH 7.8, containing 1mM dithiothreitol, 2mM EDTA, 10~M [a-
32P]GTP, and 1
,uM H-rab protein. GTPase activity is initiated by adding MgClz to a final
concentration of 10 mM.
Samples are removed at various time points, mixed with an equal volume of ice-
cold O.SmM EDTA,
and frozen. Aliquots are spotted onto polyethyleneimine-cellulose thin layer
chromatography plates,
which are developed in 1M LiCl, dried, and autoradiographed. The signal
detected is proportional to
lN'TSIG activity.
Alternatively,1NTSIG activity may be demonstrated as the ability to interact
with its
associated LMW GTPase in an i~2 vitro binding assay. The candidate LMW GTPases
are expressed
as fusion proteins with glutathione S-transferase (GST), and purified by
affinity chromatography on
glutathione-Sepharose. The LMW GTPases are loaded with GDP by incubating 20 mM
Tris buffer,
pH 8.0, 'containing 100 mM NaCl, 2 mM EDTA, 5 mM MgCla, 0.2 mM DTT, 100 ~,M
AMP-PNP and
10 ~.M GDP at 30 °C for 20 minutes. INTSIG is expressed as a FLAG
fusion protein in a baculovirus
system. Extracts of these baculovirus cells containing INTSIG-FLAG fusion
proteins are precleared
with GST beads, then incubated with GST-GTPase fusion proteins. The complexes
formed are
precipitated by glutathione-Sepharose and separated by SDS-polyacrylamide gel
electrophoresis. The
separated proteins are blotted onto nitrocellulose membranes and probed with
commercially available
anti-FLAG antibodies. INTSIG activity is proportional to the amount of INTSIG-
FLAG fusion protein
detected in the complex.
The role of INTSIG can be assayed in vitro by monitoring the mobilization of
Ca++ as part of
the signal transduction pathway. (See, e.g., Grynkievicz, G. et al. (1985) J.
Biol. Chem. 260:3440;
McColl, S. et al. (1993) J. Tmmunol. 150:4550-4555; and Aussel, C. et al.
(1988) J. Innmunol. 140:215-
220.) The assay requires preloading neutrophils or T cells with a fluorescent
dye such as FURA-2.
Upon binding Ca++, FURA-2 exhibits an absorption shift that can be observed by
scanning the
excitation spectrum between 300 and 400 nm, while monitoring the emission at S
10 nm. When the
cells are exposed to one or more activating stimuli artificially (i.e., anti-
CD3 antibody ligation of the T
cell receptor) or physiologically (i.e., by allogeneic stimulation), Ca++ flux
takes place. Ca++ flux results
from the release of Ca++from intracellular organelles or from Cap entry into
the cell through activated
Ca** channels. This flux can be observed and quantified by assaying the cells
in a fluorometer or
fluorescence activated cell sorter. Measurements of Ca++ flux are compared
between cells in their
normal state and those preloaded with 1NTSIG. Increased mobilization
attributable to increased
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INTSIG availability results in increased emission.
Another alternative assay to detect INTSIG activity is the use of a yeast two-
hybrid system
(Zalcman, G, et al. (1996) J. Biol. Chem. 271:30366-30374). Specifically, a
plasmid such as
pGAD 1318 which may contain the coding region of INTSIG can be used to
transform reporter L40
yeast cells which contain the reporter genes LacZ and IIIS3 downstream from
the binding sequences
for LexA. These yeast cells have been previously transformed with a pLexA-Rab6-
GDP (mouse)
plasmid or with a plasmid which contains pLexA-lamin C. The pLEXA-lamin C
cells serve as a
negative control. The transformed cells are plated on a histidine-free medium
and incubated at 30 °C
for 3 days. His+ colonies are subsequently patched on selective plates and
assayed for (3-
galactosidase activity by a filter assay. 1NTSIG binding with Rab6-GDP is
indicated by positive
His+/lacZ+ activity for the cells transformed with the plasmid containing the
mouse Rab6-GDP and
negative His+/lacZ+ activity for those transformed with the plasmid containing
lamin C.
Alternatively, INTSIG activity is measured by binding of 1NTSIG to a substrate
which
recognizes WD-40 repeats, such as ElonginB, by coimmunoprecipitation (Kamura,
T. et al. (1998)
Genes Dev. 12:3872-3881). Briefly, epitope tagged substrate and INTSIG are
mixed and
immunoprecipitated with commercial antibody against the substrate tag. The
reaction solution is run
on SDS-PAGE and the presence of INTSIG visualized using an antibody to the
INTSIG tag.
Substrate binding is proportional to 1NTSIG activity.
Alternatively, INTSIG activity is measured by its inclusion in coated
vesicles. INTSIG can be
expressed by transforming a mammalian cell line such as COS7, HeLa, or CHO
with a eukaryotic
expression vector encoding INTSIG. Eukaryotic expression vectors are
commercially available, and
the techniques to introduce them into cells are well known to those skilled in
the art. A small amount
of a second plasmid, which expresses any one of a number of marker genes, such
as (3-galactosidase,
is co-transformed into the cells in order to allow rapid identification of
those cells which have taken up
and expressed the foreign DNA. The cells are incubated for 48-72 hours after
transformation under
conditions appropriate for the cell line to allow expression and accumulation
of INTSIG and (3
galactosidase.
In the alternative, INTSIG activity is measured by its ability to alter
vesicle trafficking
pathways. Vesicle trafficking in cells transformed with INTSIG is examined
using fluorescence
microscopy. Antibodies specific for vesicle coat proteins or typical vesicle
trafficking substrates such
as transferrin or the mannose-6-phosphate receptor are commercially available.
Various cellular
components such as ER, Golgi bodies, peroxisomes, endosomes, lysosomes, and
the plasmalemma are
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examined. Alterations in the numbers and locations of vesicles in cells
transformed with INTSIG as
compared to control cells are characteristic of INTSIG activity. Transformed
cells are collected and
cell lysates are assayed for vesicle formation. A non-hydrolyzable form of
GTP, GTP~yS, and an ATP
regenerating system are added to the lysate and the mixture is incubated at 37
°C for 10 minutes.
Under these conditions, over 90% of the vesicles remain coated (Orci, L. et
al. (1989) Cell 56:357-
368). Transport vesicles are salt-released from the Golgi membranes, loaded
under a sucrose
gradient, centrifuged, and fractions are collected and analyzed by SDS-PAGE.
Co-localization of
1NTSIG with clathrin or COP coatamer is indicative of INTSIG activity in
vesicle formation. The
contribution of INTSIG in vesicle formation can be confirmed by incubating
lysates with antibodies
1o specific for INTSIG prior to GTPyS addition. The antibody will bind to
INTSIG and interfere with its
activity, thus preventing vesicle formation.
Alternatively,1NTSIG activity is measured by the transfer of electrons from
(and consequent
oxidation of ) NADH to cytochrome b5 when 7NTSIG is incubated together with
NADH and
cytochrome b5. The reaction is carried out in an optical cuvette containing
aliquots of INTSIG
together with 150 mM each of NADH and cytochrome b5 in 1 M Tris-acetate
buffer, pH 8.1. The
reaction is incubated at 21 ° C and the oxidation of NADH is followed
by the change in absorption at
340 nm using an ultraviolet spectrophotometer. The activity of INTSIG is
proportional to the rate of
change of absorption at 340 nm.
Alternatively,1NTSIG activity is measured by the transfer of electrons from
cytochrome c to
an electron acceptor (KCN) in the presence of a reconstituted cytochrome c
oxidase enzyme complex
containing 1NTSIG in place of COX4. The reconstituted cytochrome c oxidase is
incubated together
with cytochrome c and KCN in a suitable buffer. The reaction is carried out~in
an optical cuvette and
monitored by the change in absorption due to oxidation of cytochrome c using a
spectrophotometer.
Cytochrome c oxidase reconstituted in the absence of 1NTSIG is used as a
negative control. The
activity of INTSIG is proportional to the change in optical absorption
measured.
In another alternative, INTSIG activity is measured in the reconstituted NADH-
D complex by
the catalysis of electron transfer from NADH to decylubiquinone (DB). The
reaction contains 10
mg/mL NADH-D protein, 20 mM NADH in 50 mM tris-HCL buffer, pH 7.5, 50 mM NaCl,
and 1
mM KCN. The reaction is started by addition of DB at 2 uM and followed by the
change in
absorbance at 340 rim due to the oxidation of NADH using an ultraviolet
spectrophotometer.
NADH-D complex reconstituted in the absence of NHETP-3 is compared as a
negative control. The
activity of MITO in the reconstituted NADH-D complex is proportional to the
rate of change of
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absorbance at 340 nm.
Various modifications and variations of the described compositions, methods,
and systems of
the invention will be apparent to those skilled in the art without departing
from the scope and spirit of
the invention. It will be appreciated that the invention provides novel and
useful proteins, and their
encoding polynucleotides, which can be used in the drug discovery process, as
well as methods for
using these compositions for the detection, diagnosis, and treatment of
diseases and conditions.
Although the invention has been described in connection with certain
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments.
Nor should the description of such embodiments be considered exhaustive or
limit the invention to the
precise forms disclosed. Furthermore, elements from one embodiment can be
readily recombined with
elements from one or more other embodiments. Such combinations can form a
number of
embodiments within the scope of the invention. It is intended that the scope
of the invention be
defined by the following claims and their equivalents.
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CA 02458645 2004-02-16
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Table 5
PolynucleotideIncyte ProjectRepresentative Library
SEQ ID:
ID NO:
46 2562907CB SKIRNORO1
1
47 3744219CB LUNGDIS03
1
48 5515030CB TESTNOT 11
1 '
49 1681532CB UTREDME06
1
50 70845770CB BRAIUNFOl
1
51 3448184CB BRAWTDR02
1
52 6322968CB SMCRUNE01
1
53 6819485CB SINTFER02
1
54 7499882CB SIN'TNOROl
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